Anti-nkg2a antibodies and uses thereof

ABSTRACT

The present disclosure provides isolated monoclonal antibodies (e.g., humanized and human monoclonal antibodies), or antigen-binding fragments thereof, that specifically bind to human natural killer cell inhibitory receptor group 2A (NKG2A) protein with high affinity and exhibit therapeutically desirable functional properties, such as for the treatment of, for example, cancer. Immunoconjugates, bispecific molecules, and pharmaceutical compositions comprising the anti-NKG2A antibodies of the invention are also provided. Nucleic acid molecules encoding the antibodies, expression vectors, host cells, and methods of treatment of, for example, cancer using the antibodies are further provided. Combination therapy, in which an anti-NKG2A antibody in the present disclosure is co-administered with at least one additional agent such as another antibody (e.g., anti-PD-1, anti-PD-L1, and/or anti-CTLA-4 antibodies), is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Provisional Application No. 62/768,471, filed Nov. 16, 2018, and U.S. Provisional Application No. 62/927,211, filed Oct. 29, 2019, both which are incorporated herein by reference in their entireties.

TECHNICAL FIELD

This invention relates to anti-NKG2A (natural killer cell inhibitory receptor group 2A) antibodies and pharmaceutical compositions thereof. The invention also relates to methods for using such antibodies, including methods for treating diseases such as cancer by administering the anti-NKG2A antibodies and pharmaceutical compositions thereof.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 4, 2019, is named 13119-US-NP SL.txt and is 269,069 bytes in size.

BACKGROUND

Cancer is a global epidemic. According to the Global Health Data Exchange, cancer is one of the leading causes of disease and the second leading cause of death, causing about 17% of deaths worldwide. (“Hannah Ritchie and Max Roser, “Causes of Death—Share of deaths by cause, World, 2017”, OurWorldInData.org, 2018, available at https://ourworldindata.org/grapher/share-of-deaths-by-cause-2016). According to the World Health Organization, even in 2010, the economic impact of cancer was $1.16 trillion; in 2018, cancer accounted for an estimated 9.6 million deaths worldwide. (“Cancer.” World Health Organization. World Health Organization, 2018, available at https://www.who.int/news-room/fact-sheets/detail/cancer). According to the National Cancer Institute estimates, in 2019, over 1.7 million new patients will be diagnosed with cancer, and over 600,000 patients will die from cancer in the United States. (“Cancer Stat Facts: Cancer of Any Site.” SEER Training Modules, U. S. National Institutes of Health, National Cancer Institute, 2019 <https://seer.cancer.gov/statfacts/html/all.html>).

Traditional cancer treatments include surgery, radiation therapy, and chemotherapy, amongst other therapies. In recent years, immuno-oncology or immunotherapy has emerged as a new option to treat cancer using the body's immune system. Immuno-oncology is different from traditional cancer treatments, which, for example, have tried to target tumors directly and/or to disrupt the tumor blood supply. Instead, immuno-oncology is designed to harness the patient's own immune system to help restore or to enhance the patient's anti-tumor immune response. Without an immuno-oncology approach, a patient's own immune responses frequently fail to prevent tumor growth for various reasons. For example, many tumors have developed specialized mechanisms to evade the patient's immune responses. Tumor cells may also lose the expression of antigens that may be recognized by the patient's immune system. In other cases, a tumor's rapid growth may even overwhelm the immune system's capacity to effectively control the tumor. (Abbas et al., “Chap. 18: Immunity to Tumors”, in Cellular and Molecular Immunology, 9^(th) ed. Elsevier, Inc., (2018)). Understanding how the immune system affects cancer development and how the immune system can be used to treat cancer has presented challenging, multi-faceted problems. For example, many patients do not respond to existing immuno-oncology treatments, and some develop resistance mechanisms, such as T cell exhaustion, which is when a T cell, a specific type of white blood cell, no longer functions properly. (Dempke et al., Eur. J. of Cancer, 74: 55-72 (2017)).

Patients need improved treatments for diseases such as cancer to improve upon traditional therapies, including currently available cancer immunotherapies. There is a great need for novel immuno-oncology agents used either alone or in combination with existing agents to improve patients' response rates and to overcome drug resistance.

SUMMARY OF THE INVENTION

The present invention provides, in some aspects, isolated monoclonal antibodies (e.g., humanized and human monoclonal antibodies) that bind to human NKG2A protein (SEQ ID NO: 1), i.e., anti-NKG2A antibodies, including anti-hNKG2A antibodies that exhibit desirable functional properties. In one aspect, the desirable functional properties of the anti-NKG2A antibodies disclosed herein include to stimulate immune responses, for example, to treat cancer. In another aspect, the desirable functional properties of the anti-NKG2A antibodies disclosed herein include to treat virus-infected subjects, including human patients. In some embodiments, the anti-NKG2A antibodies disclosed herein treat infectious diseases. In another aspect, the anti-NKG2A antibodies disclosed herein treat autoimmune conditions. In other embodiments, the anti-NKG2A antibodies of the invention are used as antagonistic anti-NKG2A antibodies to stimulate and/or enhance an immune response in a subject, e.g., to stimulate and/or enhance the anti-tumor responses of the immune system, including natural killer cells and/or T cells. In other embodiments, the anti-NKG2A antibodies of the invention are used in combination with other antibodies to treat various conditions, including cancer, infectious diseases, including viral infections, and autoimmune diseases. Accordingly, in some embodiments, the anti-NKG2A antibodies disclosed herein, either alone or in combination with other treatments, such as other immuno-oncology treatments and/or chemotherapy and/or surgery, are used to treat various conditions or diseases, including cancer and viral infections. In other embodiments, the anti-NKG2A antibodies disclosed herein are used in methods to detect NKG2A protein in a sample.

In one aspect, the present invention provides an isolated monoclonal antibody, or an antigen-binding fragment thereof, wherein the antibody, or an antigen-binding fragment thereof, specifically binds human NKG2A and exhibits at least one of the following properties:

-   -   (a) reduces (e.g., blocks) binding and/or interaction of an         NKG2A ligand (e.g., HLA-E in humans) to human NKG2A protein;     -   (b) reverses NKG2A-mediated inhibitory signaling;     -   (c) does not bind, or binds with low affinity to, human NKG2C         protein;     -   (d) binds to human and/or cynomolgus NKG2A;     -   (e) enhances natural killer cell response;     -   (f) enhances the functional activity of T cells;     -   (g) has reduced binding to human Fc gamma receptor (FcγR);     -   (h) induces and/or enhances an anti-tumor immune response;     -   (i) induces and/or enhances an anti-viral immune response;         and/or     -   (j) has low immunogenicity in subjects, including human         subjects.

In one embodiment, the isolated monoclonal antibody, or an antigen-binding fragment thereof, has one or more of the following properties:

(a) has an EC₅₀ value of about 0.6 nM or lower for binding to human NKG2A protein as measured by cell binding assay;

(b) has an EC₅₀ value of about 9.0 nM or higher for binding to human NKG2C protein as measured by cell binding assay;

(c) has an EC₅₀ value for the binding to human NKG2A protein that is about 15-fold less than a second EC₅₀ value for binding to human NKG2C protein;

(d) has an IC₅₀ value of about 1.0 nM or lower for reducing the binding and/or interaction of HLA-E to human NKG2A protein as measured by cell blocking assay;

(e) binds to human NKG2A protein with a K_(D) of about 0.4 nM or lower as measured by Scatchard analysis;

(f) binds to human NKG2A with a K_(D) of about 61 nM or lower as measured by surface plasmon resonance;

(g) binds to cynomolgus NKG2A with a K_(D) of about 1.0 nM or lower as measured by Scatchard analysis;

(h) is internalized upon binding to NKG2A-expressing cells;

(i) increases interferon-gamma (IFNγ) production;

(j) exhibits internationalization with an EC₅₀ value of about 0.5 nM or lower; and/or

(k) wherein the half-life of the anti-NKG2A antibody:NKG2A complex is about 40 seconds or longer. In some embodiments, the half-life of the anti-NKG2A antibody:NKG2A complex is measured using surface plasmon resonance analysis.

In one embodiment, the anti-NKG2A antibodies, or antigen binding fragment thereof, disclosed herein reduce (e.g., block) the interaction of human NKG2A protein and human NKG2A ligand (i.e., HLA-E).

In another aspect, the present invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human NKG2A protein, wherein the antibody comprises:

(a) a heavy chain variable domain comprising amino acid sequences of SEQ ID NOs: 10, 11, 12, and a light chain variable domain comprising amino acid sequences of SEQ ID NOs: 13, 14, and 15;

(b) a heavy chain variable domain comprising amino acid sequences of SEQ ID NOs: 10, 11, and 12, and a light chain variable domain comprising amino acid sequences of SEQ ID NOs: 154, 14, and 15; or

(c) a heavy chain variable domain comprising amino acid sequences of SEQ ID NOs: 10, 11, and 12, and a light chain variable domain comprising amino acid sequences of SEQ ID NOs: 155, 14, and 15.

In another aspect, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human NKG2A protein, and comprises heavy and light chain variable regions, wherein

(a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85% at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 167; and/or

(b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85% at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 164, or SEQ ID NO: 169, respectively.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human NKG2A protein, comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 8, and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 9.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human NKG2A protein, comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 8, and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 164.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human NKG2A protein, comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 167, and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 169.

In another aspect, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human NKG2A protein, wherein the heavy and light chains consist essentially of:

(a) the amino acid sequences of SEQ ID NOs: 7 and 5, respectively;

(b) the amino acid sequences of SEQ ID NOs: 7 and 19, respectively; or

(c) the amino acid sequences of SEQ ID NOs: 35 and 36, respectively.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, competes for binding to NKG2A protein with or binds to the same epitope as the anti-NKG2A antibodies disclosed herein.

In another aspect, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that, when bound to human NKG2A protein, the antibody, or an antigen-binding fragment thereof, specifically binds to the following amino acid residues as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS):

(a) (SEQ ID NO: 156) LSIDNEEMKF; (b) (SEQ ID NO: 157) PSSWIGVFRNSSHHPW; (c) (SEQ ID NO: 158) LAFKHEIKDSDN; and (d) (SEQ ID NO: 159) QVNRLKSAQQCGSSIIYHC. In some embodiments, the monoclonal antibody blocks the binding of an NKG2A ligand (e.g., HLA-E in humans) to human NKG2A protein.

In another aspect, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that, when bound to human NKG2A, specifically binds to the following amino acid residues as determined by HDX-MS and/or fast photochemical oxidation of proteins (FPOP) epitope mapping:

(a) (SEQ ID NO: 156) LSIDNEEMKF (b) (SEQ ID NO: 157) PSSWIGVFRNSSHHPW (c) (SEQ ID NO: 158) LAFKHEIKDSDN (d) L; and (e) (SEQ ID NO: 159) QVNRLKSAQQCGSSIIYHC. In some embodiments, the antibody blocks the binding of an NKG2A ligand (e.g., HLA-E in humans) to human NKG2A protein.

In some embodiments, the isolated monoclonal antibody is a full-length antibody. In other embodiments, the isolated monoclonal antibody is a full-length IgG1 antibody. In some other embodiments, the isolated monoclonal antibody is an antibody fragment. In other embodiments, the antibody fragment is a Fab, Fab′, (Fab′)2, Fv, or scFv fragment. In other embodiments, the isolated monoclonal antibody is a human, humanized, or chimeric antibody.

In one aspect, the invention provides an isolated, full-length monoclonal antibody that binds specifically to human NKG2A protein, wherein

(a) the heavy chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and/or

(b) the light chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the heavy chain of the isolated, full-length monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 7, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 5.

In another aspect, the isolated, full-length monoclonal antibody specifically binds to human NKG2A protein, wherein the heavy chain consists essentially of the amino acid sequence set forth in SEQ ID NO: 7, and the light chain consists essentially of the amino acid sequence set forth in SEQ ID NO: 5.

In another aspect, the invention provides an isolated nucleic acid molecule that encodes the heavy chain variable region and/or light chain variable region of the antibodies or antigen binding fragments thereof described herein. In some embodiments, the nucleic acid molecule is complementary DNA (cDNA).

In another aspect, the invention provides an expression vector comprising the nucleic acid molecules described herein. In another aspect, the invention provides a host cell transformed with the expression vector described herein.

In another aspect, the invention provides an immunoconjugate that comprises the antibodies described herein linked to an agent.

In another aspect, the invention provides methods of producing the antibody comprising culturing the host cell described herein. In some embodiments, the methods further comprise recovering the antibody from the host cell.

In another aspect, the invention provides a bispecific molecule comprising the anti-NKG2A antibodies described herein linked to a second functional moiety.

In another aspect, the invention provides a composition comprising the anti-NKG2A antibodies described herein, or the bispecific molecules described herein, and a pharmaceutically acceptable carrier and/or a soluble neutral-active hyaluronidase glycoprotein. In some embodiments, the composition further comprises an additional therapeutic agent. In other embodiments, the additional therapeutic agent is an anti-PD-1 antibody, anti-PD-L1 antibody, and/or an anti-CTLA-4 antibody. In other embodiments, the anti-PD-1 antibody is nivolumab, and the anti-CTLA antibody is ipilimumab. In other embodiments, the antibodies described herein are for use as a medicament for treating cancer.

In some embodiments, the cancer that is treated using the anti-NKG2A antibodies, immunoconjugates, bispecific molecules, and compositions described herein is bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, endometrial, cervical, gastric, melanoma, renal, urothelial, glioblastoma multiform, or virus-related cancer. In other embodiments, the cancer is cervical, head and neck squamous cell carcinoma (HNSCC), pancreatic, non-small cell lung cancer-adenocarcinoma type (NSCLC-AD), non-small cell lung cancer-squamous cell type (NSCLC-SQC), gastric, melanoma, colorectal (CRC), endometrial, ovarian, renal cell carcinoma (RCC), urothelial carcinoma (UCC), breast, small cell lung, glioblastoma mulitforme, prostate cancer (also known as adenocarcinoma of the prostate or PRC), or non-Hodgkin lymphoma.

In some embodiments, the anti-NKG2A antibodies are for use in enhancing an immune response. In other embodiments, the invention provides use of the anti-NKG2A antibodies described herein in the manufacture of a medicament for treatment of cancer.

In one aspect, the invention provides methods for treating or delaying progression of cancer in a human subject comprising administering to the human subject an effective amount of the anti-NKG2A antibodies, immunoconjugates, bispecific molecules, or compositions described herein. In some embodiments, the cancer is bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, endometrial, cervical, gastric, melanoma, renal, urothelial, glioblastoma multiform, or virus-related cancer. In other embodiments, the cancer is cervical, head and neck squamous cell carcinoma (HNSCC), pancreatic, non-small cell lung cancer-adenocarcinoma type (NSCLC-AD), non-small cell lung cancer-squamous cell type (NSCLC-SQC), gastric, melanoma, colorectal (CRC), endometrial, ovarian, renal cell carcinoma (RCC), urothelial carcinoma (UCC), breast, small cell lung, glioblastoma mulitforme, prostate cancer (also known as adenocarcinoma of the prostate or PRC), or non-Hodgkin lymphoma.

In other embodiments, the methods further comprise administering one or more additional therapeutic agent(s) to the human subject. In some embodiments, the one or more additional therapeutic agent(s) is a chemotherapeutic agent, a radiotherapeutic agent, and/or an immunotherapeutic agent. In other embodiments, the one or more additional therapeutic agent(s) is an anti-PD-1 antibody, an anti-PD-L1 antibody, and/or an anti-CTLA-4 antibody. In some embodiments, the anti-PD-1 antibody is nivolumab, and the anti-CTLA-4 antibody is ipililumab.

In another aspect, the invention provides methods of stimulating an immune response in a human subject comprising administering to the human subject an effective amount of the anti-NKG2A antibodies, bispecific molecules, or compositions described herein. In some embodiments, the human subject has a tumor and an anti-tumor immune response is stimulated. In other embodiments, the human subject has a chronic viral infection and an anti-viral immune response is stimulated.

In another aspect, the invention provides methods of detecting presence of NKG2A protein in a sample comprising contacting the sample with the antibody, or antigen-binding fragment thereof, disclosed herein, under conditions that allow for formation of a complex between the antibody, or antigen-binding fragment thereof, and NKG2A protein, and detecting the formation of the complex. In some embodiments of the method, the antibody, or antigen-binding fragment thereof, forms a complex 15 times more quickly with NKG2A protein than with NKG2C protein.

Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, GenBank and other sequence entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C illustrate three methods that were used to discover and test the anti-NKG2A antibodies disclosed herein. FIG. 1A illustrates the hybridoma method and anti-NKG2A antibody discovery and development steps at a high level. FIG. 1B illustrates the antibody library generation method used for antibody discovery. FIG. 1C illustrates the single B cell cloning (SBCC) method.

FIG. 2 illustrates the mutational scan analysis used to optimize the anti-NKG2A antibodies that were discovered. Specifically, FIG. 2 shows the steps for generating variants of the 13F3.A4 I107T antibody and to characterize its binding to NKG2A protein. This analysis allowed the inventors to generate variants of the 13F3.A4 I107T antibody with improved properties, and provided a rich set of information about the effect of single amino acid substitutions on the binding of the 13F3.A4 I107T antibody to NKG2A protein. FIG. 2 discloses SEQ ID NOs: 183-190, respectively, in order of appearance from top to bottom.

FIG. 3 is an exemplary heat map that was generated using mutational scan analysis, and allows for the interpretation of the sequence-activity relationship of single amino acid substitutions. FIG. 3 discloses SEQ ID NOs: 191 (germline) and SEQ ID NO: 192 (parental).

FIG. 4 shows the CDR positions that were analyzed for the 13F3.A4 I107T anti-NKG2A antibody using mutational scan analysis. FIG. 4 discloses SEQ ID NOs: 30, 15, 192, 11, and 193, respectively, in order of appearance.

FIGS. 5A-E are the heat maps that were generated using the mutational scan analysis of 13F3.A4 I107T antibody for LCDR1 substitutions (SEQ ID NOs: 194 and 30, germline and parental, respectively) (FIG. 5A); LCDR3 substitutions (SEQ ID NOs: 195 and 15, germline and parental, respectively) (FIG. 5B); HCDR1 substitutions (SEQ ID NOs: 196 and 192, germline and parental, respectively) (FIG. 5C); HCDR2 substitutions (SEQ ID NOS: 42 and 11, germline and parental, respectively) (FIG. 5D); and HCDR3 substitutions (SEQ ID NOS: 193 and 193, germline and parental, respectively (FIG. 5E).

FIG. 6 shows the alignment of the canonical sequence of full length human NKG2A (SEQ ID NO: 182) and human NKG2C (SEQ ID NO: 3) amino acid sequences. About 76% of amino acid residues (177 out of 233 amino acid residues) are conserved, about 6% of amino acid residues (14 of 233 amino acid residues) are similar, and only about 18% of amino acid residues (42 of 233 amino acid residues) are different between the human NKG2A and human NKG2C proteins.

FIG. 7A shows the nucleotide sequence (SEQ ID NO: 166) and the amino acid sequence (SEQ ID NO: 167 without signal sequence) of the mature heavy chain variable (VH) region of the anti-NKG2A antibody 13F3.A4. The amino acid sequences of the VH CDR1 (SEQ ID NO: 27), VH CDR2 (SEQ ID NO: 28), and VH CDR3 (SEQ ID NO: 29) sequences are shown in gray boxes.

FIG. 7B shows the nucleotide sequences (SEQ ID NO: 168) and amino acid sequences (SEQ ID NO: 169) of the light chain variable region of the 13F3.A4 antibody. The amino acid sequences of the VL CDR1 (SEQ ID NO: 30), VL CDR2 (SEQ ID NO: 31), and VL CDR3 (SEQ ID NO: 32) sequences are shown in gray boxes.

FIG. 8A shows the nucleotide sequence (SEQ ID NO: 51) and the amino acid sequence (SEQ ID NO: 52) of the mature VH region of the anti-NKG2A antibody 2G6.C2. The amino acid sequences of the VH CDR1 (SEQ ID NO: 55), VH CDR2 (SEQ ID NO: 56), and VH CDR3 (SEQ ID NO: 57) sequences are shown in gray boxes.

FIG. 8B shows the nucleotide sequences (SEQ ID NO: 53) and amino acid sequences (SEQ ID NO: 54) of the mature VL region of the anti-NKG2A antibody 2G6.C2. The amino acid sequences of the VL CDR1 (SEQ ID NO: 58), VL CDR2 (SEQ ID NO: 59), and VL CDR3 (SEQ ID NO: 60) sequences are shown in gray boxes.

FIG. 9A shows the nucleotide sequence (SEQ ID NO: 170) and the amino acid sequence (SEQ ID NO: 171) of the mature heavy chain variable (VH) region of the anti-NKG2A antibody 11H9.A1. The amino acid sequences of the VH CDR1 (SEQ ID NO: 41), VH CDR2 (SEQ ID NO: 42), and VH CDR3 (SEQ ID NO: 43) sequences are shown in gray boxes.

FIG. 9B shows the nucleotide sequences (SEQ ID NO: 172) and amino acid sequences (SEQ ID NO: 173) of the light chain variable region of the mature anti-NKG2A antibody 11H9.A1. The amino acid sequences of the VL CDR1 (SEQ ID NO: 44), VL CDR2 (SEQ ID NO: 45), and VL CDR3 (SEQ ID NO: 46) sequences are shown in gray boxes.

FIG. 10A shows the nucleotide sequence (SEQ ID NO: 174) and the amino acid sequence (SEQ ID NO: 175) of the mature VH region of the anti-NKG2A antibody 4G5.D1. The amino acid sequences of the VH CDR1 (SEQ ID NO: 69), VH CDR2 (SEQ ID NO: 70), and VH CDR3 (SEQ ID NO: 71) sequences are shown in gray boxes.

FIG. 10B shows the nucleotide sequence (SEQ ID NO: 176) and amino acid sequences (SEQ ID NO: 177) of the VL of the 4G5.D1 antibody. The amino acid sequences of the VL CDR1 (SEQ ID NO: 72), VL CDR2 (SEQ ID NO: 73), and VL CDR3 (SEQ ID NO: 74) sequences are shown in gray boxes.

FIG. 11A shows the nucleotide sequence (SEQ ID NO: 178) and the amino acid sequence (SEQ ID NO: 179) of the mature VH region of the anti-NKG2A antibody 1G5.B2. The amino acid sequences of the VH CDR1 (SEQ ID NO: 83), VH CDR2 (SEQ ID NO: 84), and VH CDR3 (SEQ ID NO: 85) sequences are shown in gray boxes.

FIG. 11B shows the nucleotide sequence (SEQ ID NO: 180) and the amino acid sequence (SEQ ID NO: 181) of the VL region of the 1G5.B2 antibody. The amino acid sequences of the VL CDR1 (SEQ ID NO: 86), VL CDR2 (SEQ ID NO: 87), and VL CDR3 (SEQ ID NO: 88) sequences are shown in gray boxes.

FIG. 12 shows the amino acid sequence liabilities that were assessed in the 13F3.A4 antibody. The V, D, and J germline derivations are indicated. The amino acid sequences of the heavy chain variable region of the 13F3.A4 antibody (SEQ ID NO: 167) are shown on the left, and the amino acid sequences of the light chain variable region of the 13F3.A4 antibody (SEQ ID NO: 169) are shown on the right. The amino acid sequences of the VL CDR1 (SEQ ID NO: 30), VL CDR2 (SEQ ID NO: 31), and VL CDR3 (SEQ ID NO: 32) sequences are underlined. The amino acid sequences of the VH CDR1 (SEQ ID NO: 27), VH CDR2 (SEQ ID NO: 28), and VH CDR3 (SEQ ID NO: 29) sequences are underlined. The sequence liabilities that were assessed are circled and labeled.

FIG. 13 shows the full length amino acid sequence of anti-NKG2A antibody NKG2A.9. The light chain amino acid sequence is set forth in SEQ ID NO: 5, and the heavy chain amino acid sequence is set forth in SEQ ID NO: 163 (shown with the terminal lysine, which in another embodiment is absent). FIG. 13 identifies with an oval two mutations made to the 13F3.A4 VL sequence (N30S) and VH sequence (I107T) to result in the NKG2A.9 sequence. The N-glycosylation motif is shown with a dashed oval. Three mutations (L234A, L235E, and G273A) that were made to the Fc region are shown in bold italics. The terminal amino acid lysine in the heavy chain sequence was removed from the mature sequence. The amino acid sequences of the VL CDR1 (SEQ ID NO: 13), VL CDR2 (SEQ ID NO: 14), and VL CDR3 (SEQ ID NO: 15) sequences are in bold and underlined. The amino acid sequences of the VH CDR1 (SEQ ID NO: 10), VH CDR2 (SEQ ID NO: 11), and VH CDR3 (SEQ ID NO: 12) sequences are in bold and underlined.

FIG. 14 shows the full length amino acid sequence of anti-NKG2A antibody NKG2A.9. The light chain amino acid sequence is set forth in SEQ ID NO: 5, and the heavy chain amino acid sequence is set forth in SEQ ID NO: 7.

FIG. 15 shows the full length amino acid sequence of anti-NKG2A antibody NKG2A.11. The light chain amino acid sequence is set forth in SEQ ID NO: 19, and the heavy chain amino acid sequence is set forth in SEQ ID NO: 7.

FIG. 16 shows a circle plot of epitope binning results of exemplary antibodies generated by the single B cell cloning method (SBCC), and shows the diversity of these antibodies. The antibodies that cross-block each other are connected by a line. Sample antibodies that have a similar blocking profile as compared to the benchmark antibodies (13F3.A4, Z270, RD-ahNKG2a (clone 131411, Catalog No. MAB1059), and RD-ahCD94 (clone 131412, Catalog No. MAB1058)) are grouped together into groups 1-4, 6-7, and 9. Benchmark antibodies with similar blocking profiles as the sample antibody sets are also grouped together into groups 5, 8 and 10.

FIG. 17A shows the assay method to assess the ability of anti-human NKG2A antibodies generated using the SBCC method to block NKG2A/HLA-E interaction. FIG. 17 B shows that the sample antibodies only partially blocked or did not block the NKG2A/HLA-E interaction, as compared to the positive control (NKG2A.9 antibody) and negative control (isotype).

FIG. 18A-C show the binding assay method (FIG. 18A) to assess binding of anti-NKG2A antibodies to human NK2GA-expressing (FIG. 18B) and NKG2C-expressing CHO cells (FIG. 18C). As shown in FIG. 18B, the 13F3.A4, 11H9.A1, and 2EB.B1 antibodies showed specific binding to the human NKG2A-expressing CHO cells. FIG. 18D-F show the blocking assay method (FIG. 18D) used to assess blocking of anti-NKG2A antibodies of the NKG2A/HLA-E interaction (FIG. 18E) and NKG2C/HLA-E interaction (FIG. 18F). The 13F3.A4 and 11H9.A1 antibodies showed specific binding to human NKG2A-expressing CHO cells (as shown in FIG. 18E), and did not block the NKG2C/HLA-E interaction (as shown in FIG. 18F).

FIG. 19A-B illustrates the binding assay method (FIG. 19A) used, which assessed the ability of anti-NKG2A antibodies to bind to human NKG2A+ natural killer cell line (NKL)(as shown in FIG. 19B). FIG. 19C-D illustrates the blocking assay method (FIG. 19C) used, which showed that the anti-NKG2A antibodies tested blocked HLA-E binding to human NKG2A expressing NKL cells, as shown in FIG. 19D.

FIG. 20A-C illustrates the blocking assay method (FIG. 20A) used, which showed that the anti-human NKG2A antibodies tested blocked the NKG2A/HLA-E interaction in hNKG2A-expressing CHO cells, as shown in FIG. 20B-C.

FIG. 21A-C illustrates the binding assay method (FIG. 21A) used, which showed that the anti-NKG2A antibodies tested bound to human NKG2A-expressing CHO cells, as shown in FIG. 21B-C.

FIG. 22A-C illustrates the binding assay method (FIG. 22A) used, which assessed the ability of anti-NKG2A antibodies to bind to cynomolgus NKG2A-expressing CHO cells (results shown in FIG. 22B-C).

FIG. 23A-B illustrate the blocking assay method (FIG. 23A) used to assess ability of anti-NKG2A antibodies to desirably block the NKG2A/HLA-E interaction (FIG. 23B). FIG. 23C-D show the binding assay method used (FIG. 23C) that showed the ability of anti-NKG2A antibodies to bind to NKG2A-expressing CHO cells (FIG. 23D).

FIG. 24A-B show the binding assay method (FIG. 24A) used to assess whether anti-NKG2A antibodies bound to cynomolgus NKG2A+NKLs (FIG. 24B). As shown in FIG. 24B, the 11H9.A1 and 4G5.D1 antibodies did not bind to cynomolgus NKG2A+ NK cells, while the 13F3.A4 antibody desirably bound to cynomolgus monkey NKG2A+ NK cells, as indicated by the EC₅₀ value of 0.2 nM.

FIG. 25A illustrates the in vitro method used to assess whether anti-NKGA antibodies increased NK cell degranulation. FIG. 25B-C are graphs of the flow cytometry analysis results, and shows that all tested anti-NKG2A antibodies (the NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, NKG2A.15, NKG2A.5, 2G6.C2, and 4G5.D1 antibodies) enhanced NK cell degranulation (as measured by fold change in % CD107a compared to the isotype control) among NKG2A+ NK cells. The 25E7.G8 clone (as shown in FIG. 25B) is a non-blocking antibody that was used as a negative control.

FIG. 26A illustrates the in vitro experiments using NKL cells and CHO/MICA/HLA-E to show that the anti-NKG2A antibodies blocked the NKG2A/HLA-E interaction among activated NK cells and increased IFN-γ production. FIG. 26B shows that the 13F3.A4, 11H9.A1, and 4G5.D1 antibodies desirably increased IFN-γ production as compared to the isotype control (human IgG1.3 antibody). The 25E7.G8 clone was used as a negative control.

FIG. 27A illustrates the in vitro experiments used to assess whether anti-NKG2A antibodies enhanced the CD8+ T cell response of increased IFNγ in Hs766T target cells, a pancreatic carcinoma cell line. FIG. 27B is a graphical representation of the assay results and showed that the 13F3.A4 and 11H9.A1 antibodies increased IFN-γ production compared to the isotype control. The 25E7.G8 clone was used as a negative control.

FIG. 28A-B show the results of differential hydrogen-deuterium exchange (HDX) analysis of mFc-hNKG2A-hCD94 upon interaction with NKG2A.9 antibody (FIG. 28A) and with 13F3.A4 antibody (FIG. 28B), respectively. The epitope sequences are labeled in FIGS. 28A-B (SEQ ID NOS: 119-122, disclosed from left to right along the analysis).

FIG. 29A-B show FPOP protection percentages for four residues (M163, F179, H184, L206) in hNKG2A upon interaction with NKG2A.9 antibody (FIG. 29A) and with 13F3.A4 antibody (FIG. 29B), respectively.

FIG. 30 shows the epitopes of NKG2A.9 and 13F3.A4 antibodies as determined by HDX-MS and by FPOP mapped to the mFc-hNKG2A-hCD94 sequence (SEQ ID NO: 125). The bold, underlined epitopes were determined by HDX-MS, and the circled epitopes were determined by FPOP analysis.

FIG. 31 shows the anti-NKG2A antibody epitope (for example, of the NKG2A.9 and 13F3.A4 antibodies) visualized on the NKG2A/CD94/HLA-E crystal structure. The anti-NKG2A antibody epitope is shown in black.

FIG. 32A-B show the alignment of VH region (SEQ ID NOS: 167, 8, and 8, respectively, in order of appearance from top to bottom) (FIG. 32A) and VL regions (SEQ ID NOS: 169, 9, and 164, respectively, in order of appearance from top of bottom) (FIG. 32B) of certain portions of the anti-NKG2A antibodies (13F3.A4, NKG2A.9, and NKG2A.11). This alignment resulted in the discovery of anti-NKG2A antibodies with consensus CDR sequences shown in boxes.

FIG. 33 shows assay results that the NKG2A.9 antibody, as compared to isotype, reversed the inhibition of NK-κB signaling in NKG2A-expressing Jurkat T cells stimulated by CHO/scOKT3/HLA-E.

FIG. 34A illustrates the experimental method used to analyze the effect of NKG2A.9 and anti-PD-L1 antibodies, either alone or in combination, on IFN-γ induction by NKG2A⁺ CD8+ T cells isolated from healthy peripheral blood mononuclear cells (PBMCs). FIG. 34B shows the results of the method in which the combination of NKG2A.9 and anti-PD-L1 antibodies enhanced IFN-γ production by NKG2A⁺ CD8+ T cells in a dose-dependent manner.

FIG. 35A-B illustrates the assay method (FIG. 35A) used to show that NKG2A.9 and/or anti-PD-L1 antibodies enhanced IFN-γ production in NKG2A⁺ CD8+ T cells isolated from human tumors co-cultured with CHO/scOKT3/HLA-E/PD-L1 (FIG. 35B).

FIG. 36A is a graph showing that the NKG2A.9 antibody was internalized after binding to NKG2A-expressing cells. FIG. 36B is a graph showing the binding kinetics of the NKG2A.9 antibody and demonstrates that the NKG2A.9 antibody was internalized in a dose-dependent manner with an EC₅₀ of 0.5 nM.

FIG. 37A-B illustrate the method used (FIG. 37A) to show that the NKG2A.9 antibody, as compared to isotype, increased NK cell degranulation in a dose-dependent manner, as measured by % CD107a expression measured by flow cytometry. FIG. 37C-D illustrates the experimental method used (FIG. 37C) that showed that the NKG2A.9 antibody, as compared to isotype, increased lysis of HLA-E-expressing tumor cells in a dose-dependent manner (FIG. 37D).

FIG. 38A illustrates the method used to measure the effect of the 13F3.A4 antibody on IFN-γ production in NKLs co-cultured with CHO/MICA/HLA-E. FIG. 38B shows that the 13F3.A4 antibody increased IFN-γ production in NKLs as compared to the isotype.

FIG. 39 shows the results of a dose titration study in which 10 mg/kg, 3 mg/kg, and 1 mg/kg of an anti-mNKG2A antibody (NKG2A.3) was administered as a monotherapy, and inhibited tumor growth in a colon carcinoma tumor model with a 48%, 56%, and 30% reduction in mean tumor volume, respectively. No efficacy was observed for the 0.3 mg/kg dose.

FIG. 40A-E show the results of in vivo studies in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in mouse models where anti-mNKG2A antibody monotherapy showed single-agent activity in the CT26 colorectal tumor mouse model. FIG. 40A-D show the tumor volume at various time points after tumor implantation in mice (n=10/group) treated with isotype (FIG. 40A), anti-mNKG2A antibody alone (FIG. 40B), anti-mPD-1 antibody alone (FIG. 40C), or a combination of anti-mNKG2A and anti-mPD1 antibodies (FIG. 40D). FIG. 40E shows the average tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mNKG2A antibody alone, anti-mPD-1 antibody alone, or a combination of anti-mNKG2A and anti-mPD-1 antibodies.

FIG. 41A-C are graphs showing the results of in vivo studies in which anti-NKG2A and anti-PD-1 antibodies increased NK (FIG. 41A) and tumor-specific CD8+ T cell cytotoxicity and IFN-γ in a murine colon carcinoma model (FIG. 41B-C).

FIG. 42A-E shows the anti-tumor activity of anti-mNKG2A and anti-mCTLA-4 antibodies, either alone or in combination, in the 1956 mouse sarcoma model. FIG. 42A-D show the tumor volume at various time points after tumor implantation in mice treated with isotype (FIG. 42A), anti-mCTLA-4 antibody (FIG. 42B, CTLA-4 IgG2a, 0.1 mg/kg), anti-mNKG2A antibody (FIG. 42C, 10 mg/kg), or a combination of anti-mNKG2A and anti-mCTLA-4 (FIG. 42D). FIG. 42E shows the average tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mCTLA-4 alone, anti-mNKG2A alone, or combination of anti-mNKG2A and anti-mCTLA-4.

FIG. 43 shows anti-tumor activity of anti-NKG2A, anti-PD-1, and anti-LAG3 antibodies and combinations thereof in a murine lymphoma model. Administering anti-NKG2A antibody alone had a survival benefit, with a 10% survival rate. Combination therapy of anti-NKG2A antibody with either anti-mPD-1 or anti-mLAG-3 antibodies extended the survival rate to 50% and 70%, respectively. The triple combination of anti-mNKG2A, anti-mPD-1, and anti-mLAG-3 antibodies provided the greatest benefit with an 80% survival rate.

FIG. 44 shows that after treatment with anti-mNKG2A antibody in a murine CT26 colon carcinoma model, the NKG2A expression level was reduced on both splenic and tumor infiltrating lymphocyte (TIL) NK cells as compared with the isotype.

FIG. 45 shows the results of NKG2A expression as evaluated by immunohistochemistry on different tumor types.

FIG. 46 shows the binding profile of the FITC-conjugated NKG2A.6 antibody in multiple tumors.

FIG. 47 shows the results of HLA-E expression in 7 different tumor types as evaluated by immunohistochemistry.

FIG. 48 shows representative images of HLA-E expression in different tumor types as evaluated by immunohistochemistry.

FIG. 49A-B show levels of soluble HLA-E levels across healthy control patients and cancer patients.

FIG. 50 shows the clinical development plan for anti-NKG2A antibodies discussed herein, including patient selections steps and various combination therapies with the anti-NKG2A antibodies described herein.

FIG. 51A-D are graphs showing the kinetics and binding affinity of the NKG2A.9 antibody as determined by Biacore analysis.

FIG. 52A-B shows the kinetic and binding affinities of NKG2A.9 as measured by Scatchard analysis.

FIG. 53 shows the results of NK degranulation assay comparing P1-069366 to NKG2A.9 and isotype. The NKG2A.9 antibody showed functionality in the NK degranulation assay, while the P1-069366 antibody did not.

FIG. 54A-C show the results of using an in silico HLA binding tool to analyze the 13F3.A4 antibody for undesirable binding clusters. FIGS. 54A-C disclose SEQ ID NOS: 197-204, respectively, in order of appearance from top to bottom.

FIG. 55 show the results of an in vitro DC:T cell proliferation assay in demonstrating the low immunogenicity risk of anti-NKG2A antibodies, specifically, the NKG2A.6, NKG2A.9, and NKG2A.11 antibodies.

FIG. 56A-D show the binding affinity of the NKG2A.9 antibody to human NKG2A-CD94 heterodimers (FIG. 56A-B) and NKG2C-CD94 heterodimers (FIG. 56C-D) at 37° C. as determined by Biacore using both single-cycle kinetics (FIGS. 56A and 56C) and multi-cycle kinetics (FIGS. 56B and 56D). The SPR response is shown as a function of analyte association and dissociation.

FIG. 57 shows a boxplot of total HLA-E positivity score across 16 different tumor types as evaluated by immunohistochemistry. Total HLA-E score is defined as percent of combined cytoplasmic and/or membrane HLA-E positivity in the tumor cells.

DETAILED DESCRIPTION

In some aspects, the present invention provides isolated antibodies, such as monoclonal antibodies, e.g., humanized, human, and chimeric monoclonal antibodies, that specifically bind to human NKG2A (“hNKG2A”) and have antagonistic activity to stimulate an anti-tumor immune response. In some embodiments, the anti-NKG2A antibodies described herein comprise particular structural features such as CDR regions comprising particular amino acid sequences. In other embodiments, the anti-NKG2A antibodies compete for binding to human NKG2A protein with, or bind to the same or similar epitope as, the anti-NKG2A antibodies of the present invention.

In some aspects, the present invention provides methods of making such anti-NKG2A antibodies, immunoconjugates, and bispecific molecules comprising such anti-NKG2A antibodies or antigen-binding fragments thereof, and pharmaceutical compositions formulated to contain the anti-NKG2A antibodies or antigen-binding fragments thereof. In some aspects, the present invention provides methods of using the anti-NKG2A antibodies, either alone or in combination with other agents, e.g., other immuno-oncology agents (e.g., antibodies), chemotherapy, radiation therapy, and/or surgery, to enhance the immune response. Accordingly, in some embodiments, the anti-NKG2A antibodies described herein are used to treat a variety of conditions, including, for example, to safely and effectively treat cancer and/or infections.

An important role of the immune system is its ability to differentiate between normal cells and “foreign” cells. The immune system can thus attack the foreign cells and leave normal cells alone. Tumors express antigens that are recognized as foreign by the host. The immune system uses “checkpoints,” which are molecules on certain immune cells that need to be activated or inactivated to begin an immune response. Tumor cells can sometimes use these checkpoints to avoid being attacked by the immune system. Some immuno-oncology drugs target these checkpoints by acting as checkpoint inhibitors. Programmed death protein 1 (PD-1) is a checkpoint inhibitor that acts as a brake to prevent T cells from attacking other cells in the body. PD-1 does this when it binds to programmed death ligand 1 (PD-L1), a protein on some normal (and cancer) cells. When PD-1 binds to PD-L1, this interaction signals the T cell to not attack other cells. Some cancer cells have large amounts of PD-L1, which helps them evade immune attack. Therapeutic agents such as monoclonal antibodies that target this PD-1/PD-L1 interaction, such as nivolumab (Opdivo®), can block the PD-1/PD-L1 binding to increase the body's immune response against tumor cells.

Natural killer cell inhibitory receptor Group 2A (NKG2A) is a member of the NKG2 lectin receptor family that also includes NKG2C, NKG2D, and NKG2E. (Iwaszko and Bogunia-Kubik, Arch Immunol Ther Exp, 59:353-67 (2011)). NKG2A, NKG2C, and NKG2E have high homology in their amino acid sequence of the extracellular domains, whereas NKG2D is a functionally distinct type of receptor. NKG2A forms a heterodimer with CD94. Among NKG2/CD94 heterodimers, NKG2A/CD94 is the only receptor that has inhibitory function, whereas NKG2C/CD94 and NKG2E/CD94 are activating receptors. NKG2D is also an activating receptor, but it does not form a heterodimer with CD94, nor does NKG2D bind HLA-E. The NKG2/CD94 receptors recognize non-classical major histocompatibility (MHC) class I molecules, which is Human Leukocyte Antigen-E (HLA-E) in humans and Qa-1 in mice. (Braud et al, Nature, 391:795-99 (1998), Vance et al., J. Exp. Med. 188:1841-48 (1998). NKG2A/CD94 binds to HLA-E with about a six-fold stronger affinity than NKG2C/CD94. Id. NKG2A is expressed on natural killer (NK), effector/memory CD8₊ T, NKT, and gamma delta (γδ) T cells. NKG2A expression is induced by T cell receptor (TCR) engagement and after stimulation with certain cytokines, including IL-2, IL-10, IL-15, IL-18, IL-21, although the capacity of cytokines to induce NKG2A expression depends on TCR engagement (Cho, Blood, 118:116-28 (2011). NKG2A has two immunoreceptor tyrosine-based inhibitory motifs (ITIMS) that transmit intracellular inhibitory signals. (Kabat et al, J. Immunol, 169:1948-58 (2002); Le Dréan El, Eur. J. Immunol 28:264076 (1998). The anti-NKG2A antibodies described herein inhibit the NKG2A protein and thus act as checkpoint inhibitors.

Definitions

In order that the present description may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. Additional definitions are set forth throughout the detailed description. The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

As used herein, “NKG2A” refers to the natural killer cell inhibitory receptor Group 2 protein that in humans is encoded by the NKG2A gene. NKG2A is also known as, for example, CD159 antigen-like family member A, NK cell receptor A, NKG2A-activating NK receptor, NKG2-AB-activating NK receptor, killer cell lectin like receptor C1 (CD159a), and NKG2-A/NKG2-B type II integral membrane protein.

Three isoforms of human NKG2A protein that correspond to five variants of the mRNA transcript have been identified.

Isoform 1 that corresponds to variant 1 (nucleotide sequence set forth at SEQ ID NO: 1 or 209, and amino acid sequence set forth at SEQ ID NO: 2) and variant 3 (nucleotide sequence set forth at SEQ ID NO: 212, and amino acid sequence set forth at SEQ ID NO: 182) consists of 233 amino acids and represents the canonical NGK2A sequence. Isoform 1 variant 3 is also a naturally occurring variant that is a Single Nucleotide Polymorphism (SNP) at residue 29 in which asparagine (N) is changed to a serine (S), i.e., N29S (SEQ ID NO: 182).

Isoform 2 that corresponds to variant 2 (nucleotide sequence set forth at SEQ ID NO: 210, and amino acid sequence set forth at SEQ ID NO: 206) and variant 4 (nucleotide sequence set forth at SEQ ID NO: 211, and amino acid sequence set forth at SEQ ID NO: 207) lacks an in-frame coding exon resulting in the absence of residues 96-113 compared to variant 1 and variant 3, respectively, and is also referred to as NKG2A isoform NKG2-B. Variant 4 has the N29S SNP.

Isoform 3 that correspond to variant 5 (nucleotide sequence set forth at SEQ ID NO: 208, and amino acid sequence set forth at SEQ ID NO: 205) consists of 228 amino acids, and lacks residues 229-233 that encode five C-terminal amino acids. This variant also has the N29S SNP.

Below are the amino acid sequences of the known human NGK2A variants.

(1) Variant 1: Human NGK2A isoform 1 (nucleotide sequence set forth at SEQ ID NO: 1 or 209 (Accession No. NM 002259.5), and amino acid sequence set forth at SEQ ID NO: 2 (Accession No. NP 002250.2; UniProt ID P26715-1)):

(SEQ ID NO: 2) MDNQGVIYSD LNLPPNPKRQ QRKPKGNKNS ILATEQEITY AELNLQKASQ  50 DFQGNDKTYH CKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSTLIQR 100 HNNSSLNTRT QKARHCGHCP EEWITYSNSC YYIGKERRTW EESLLACTSK 150 NSSLLSIDNE EEMKFLSIIS PSSWIGVFRN SSHHPWVTMN GLAFKHEIKD 200 SDNAELNCAV LQVNRLKSAQ CGSSIIYHCK HKL 233 (2) Variant 2: Human NGK2A isoform 2 is also referred to as NKG2A isoform NKG2B (nucleotide sequence set forth at SEQ ID NO: 210 (Accession No. NM 007328.4), and amino acid sequence set forth at SEQ ID NO: 206 (Accession No. NP 015567.2; UniProt ID P26715-2)):

(SEQ ID NO: 206) MDNQGVIYSD LNLPPNPKRQ QRKPKGNKNS ILATEQEITY AELNLQKASQ  50 DFQGNDKTYH CKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSRHCGH 100 CPEEWITYSN SCYYIGKERR TWEESLLACT SKNSSLLSID NEEEMKFLSI 150 ISPSSWIGVF RNSSHHPWVT MNGLAFKHEI KDSDNAELNC AVLQVNRLKS 200 AQCGSSIIYH CKHKL 215 (3) Variant 3: Human NGK2A isoform 1 with the N29S SNP bolded and highlighted (nucleotide sequence set forth at SEQ ID NO: 212 (Accession No. NM 213658.2), and amino acid sequence set forth at SEQ ID NO: 182 (Accession No. NP 998823.1 or AAL65234.1):

(SEQ ID NO: 182) MDNQGVIYSD LNLPPNPKRQ QRKPKGNK S S ILATEQEITY AELNLQKASQ  50 DFQGNDKTYH CKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSTLIQR 100 HNNSSLNTRT QKARHCGHCP EEWITYSNSC YYIGKERRTW EESLLACTSK 150 NSSLLSIDNE EEMKFLSIIS PSSWIGVFRN SSHHPWVTMN GLAFKHEIKD 200 SDNAELNCAV LQVNRLKSAQ CGSSIIYHCK HKL 233 (4) Variant 4: Human NKG2A isoform 2 also corresponds to variant 4 (nucleotide sequence set forth at SEQ ID NO: 211 (Accession No. NM 213657.2), and amino acid sequence set forth at SEQ ID NO: 207 (Accession No. NP 998822.1)):

(SEQ ID NO: 207) MDNQGVIYSD LNLPPNPKRQ QRKPKGNK S S ILATEQEITY AELNLQKASQ  50 DFQGNDKTYH CKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSRHCGH 100 CPEEWITYSN SCYYIGKERR TWEESLLACT SKNSSLLSID NEEEMKFLSI 150 ISPSSWIGVF RNSSHHPWVT MNGLAFKHEI KDSDNAELNC AVLQVNRLKS 200 AQCGSSIIYH CKHKL 215 (5) Variant 5: Human NGK2A isoform 3 is also referred to as NKG2A isoform C (nucleotide sequence set forth at SEQ ID NO: 208 (Accession No. NM 001304448.1), and amino acid sequence set forth at SEQ ID NO: 205 (Accession No. NM 001291377.1)):

(SEQ ID NO: 205) MDNQGVIYSD LNLPPNPKRQ QRKPKGNK S S ILATEQEITY AELNLQKASQ  50 DFQGNDKTYH CKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSTLIQR 100 HNNSSLNTRT QKARHCGHCP EEWITYSNSC YYIGKERRTW EESLLACTSK 150 NSSLLSIDNE EEMKFLSIIS PSSWIGVFRN SSHHPWVTMN GLAFKHEIKD 200 SDNAELNCAV LQVNRLKSAQ CGSSIIYH 228 The Table below provides a summary of the DNA and protein accession numbers and corresponding SEQ ID NOs described above.

DNA Accession No. (GenBank Corresponding Protein Accession No. NKG2A SEQ unless otherwise Protein SEO ID (GenBank unless Variant ID NO specified NO otherwise specified) 1 1 or NM 002259.5 2 NP 002250.2; UniProt 209 ID P26715-1 2 210 NM 007328.4 206 NP 015567.2; UnitProt ID P26715-2 3 212 NM 213658.2 182 NP 998823.1; AAL65234.1 4 211 NM 213657.2 207 NP 998822.1 5 208 NM 001304448.1 205 NP 001291377.1

The term “antibody” or “immunoglobulin,” which is used interchangeably herein, refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In certain antibodies, e.g., naturally occurring IgG antibodies, the heavy chain constant region is comprised of a hinge and three domains, CH1, CH2 and CH3. In certain antibodies, e.g., naturally occurring IgG antibodies, each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. A heavy chain may or may not have the C-terminal lysine. Unless specified otherwise herein, the amino acids in the variable regions are numbered using the Kabat numbering system and those in the constant regions are numbered using the EU system. An immunoglobulin can be from any of the known isotypes, including IgA, secretory IgA, IgD, IgE, IgG, and IgM. The IgG isotype is divided in subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In certain embodiments, the anti-NKG2A antibodies described herein are of the IgG1 subtype. Immunoglobulins, e.g., IgG1, exist in several allotypes, which differ from each other in at most a few amino acids. “Antibody” includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies and wholly synthetic antibodies.

As used herein, an “IgG antibody” has the structure of a naturally occurring IgG antibody, i.e., it has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an anti-NKG2A IgG1, IgG2, IgG3, or IgG4 antibody consists of two heavy chains (HCs) and two light chains (LCs), wherein the two heavy chains and light chains are linked by the same number and location of disulfide bridges that occur in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bonds).

An “antigen” is a molecule or substance that triggers an immune response and to which an antibody binds. Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹¹M or less, but does not bind with high affinity to unrelated antigens. Any K_(D) greater than about 10⁻⁴ M is generally considered to indicate nonspecific binding. As used herein, an antibody that “binds specifically” to an antigen refers to an antibody that binds to the antigen and, in some cases, substantially identical antigens, with high affinity, which means having a K_(D) of 10⁻⁶ M or lower, K_(D) of 10⁻⁷ M or lower, 10⁻⁸ M or lower, 10⁻⁹ M or lower, or between 10⁻⁸ M and 10⁻¹⁰ M or lower, but does not bind with high affinity to unrelated antigens. An antigen is “substantially identical” to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of the given antigen. By way of example, an antibody that binds specifically to human NKG2A, in some embodiments, also cross-reacts with NKG2A antigens from certain non-human primate species (e.g., cynomolgus monkey NKG2A), but does not cross-react with NKG2A antigens from other species or with an antigen other than NKG2A.

As used herein, the term “antigen-binding portion” or “antigen-binding fragment” of an antibody are used interchangeably herein, and refers to one or more parts of an antibody that retain the ability to specifically bind to an antigen (e.g., human NKG2A). It has been shown that the antigen-binding function of an antibody can be performed by fragments or portions of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” or “antigen-binding fragment” of an antibody, e.g., an anti-NKG2A antibody described herein, include:

(1) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC and CH1 domains;

(2) a F(ab′)2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;

(3) a Fd fragment consisting of the VH and CH1 domains;

(4) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody,

(5) a single domain antibody (dAb) fragment (Ward et al., (1989) Nature 341:544-46), which consists of a VH domain;

(6) an isolated complementarity determining region (CDR); and

(7) a combination of two or more isolated CDRs, which can optionally be joined by a synthetic linker.

Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term “antigen-binding portion” or “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

A “bispecific” or “bifunctional antibody” is an artificial hybrid antibody having two different binding specificities, e.g., two different heavy/light chain pairs, giving rise to two antigen binding sites with specificity for different antigens. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are substantially similar and bind the same epitope(s) (e.g., the antibodies display a single binding specificity and affinity), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. The modifier “monoclonal” indicates the character of the antibody as having been obtained from a substantially homogenous population of antibodies, and does not require production of the antibody by any particular method. The term “human monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies that displays a single binding specificity and that has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by using a hybridoma method. Using the hybridoma method, a transgenic non-human animal, e.g., a transgenic mouse, is exposed to an antigen and a white blood cell known as a B cell produces antibodies that bind to the antigen, which is harvested from the transgenic non-human animal. The isolated B cells are fused with an immortalized cell to produce a hybrid cell line called a hybridoma. In one embodiment, the hybridoma has a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

As used herein, the term “recombinant human antibody” includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (1) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom; (2) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma; (3) antibodies isolated from a recombinant, combinatorial human antibody library; and (4) antibodies prepared, expressed, created, or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies comprise variable and constant regions that use particular human germline immunoglobulin sequences are encoded by the germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region contains the antigen binding domain, which is encoded by various genes that rearrange to form an antibody specific for a foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase antibody affinity to the foreign antigen. The constant region will change in further response to an antigen (i.e., isotype switch). Thus, the rearranged and somatically mutated nucleic acid molecules that encode the light chain and heavy chain immunoglobulin polypeptides in response to an antigen cannot have sequence identity with the original nucleic acid molecules, but instead will be substantially identical or similar (e.g., have at least 80% identity).

As used herein, a “human antibody” refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The anti-NKG2A antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., because of mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another non-human mammalian species, such as a mouse, have been grafted onto human framework sequences. As used herein, the terms “human” and “fully human” antibodies are used interchangeably.

A “humanized antibody” refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human antibodies. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from human antibodies, whereas some, most, or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not prevent the antibody from binding to a particular antigen. A “humanized antibody” retains an antigenic specificity similar to that of the original antibody.

A “chimeric antibody” refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

As used herein, “isotype” refers to the antibody class (e.g., IgG (including IgG1, IgG2, IgG3, and IgG4), IgM, IgA (including IgA1 and IgA2), IgD, and IgE antibody) that is encoded by the heavy chain constant region genes of the antibody.

“Allotype” refers to naturally occurring variants within a specific isotype group, where variants differ in a few amino acids. (See, e.g., Jefferis et al. (2009) mAbs 1:1). Anti-NKG2A antibodies described herein can be of any allotype. As used herein, antibodies referred to as “IgG1F,” “IgG1.1f,” or “IgG1.3f” isotype are IgG1, effectorless IgG1.1, or effectorless IgG1.3 antibodies of the allotype “f,” respectively.

The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the phrase “an antibody that binds specifically to an antigen.”

As used herein, an “isolated antibody” refers to an antibody that is substantially free of other proteins and cellular materials.

As used herein, an “effector function” refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event that results therefrom. Exemplary “effector functions” include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody dependent cell-mediated phagocytosis (ADCP), and downregulation of a cell surface receptor (e.g., the B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).

An “Fc receptor” or “FcR” is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to an IgG antibody comprise receptors of the FcγR family, including allelic variants and alternatively spliced forms of these receptors. The FcγR family consists of three activating (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIb, or equivalently FcγRIIB) receptor. Various exemplary properties of human FcγRs are known in the art. The majority of innate effector cell types co-express one or more activating FcγR and the inhibitory FcγRIIb, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but not the inhibitory FcγRIIb in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the types of activating Fc receptors that it binds to.

As used herein, an “Fc region” (fragment crystallizable region) or “Fc domain” or “Fc” refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Thus, an Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments, derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains. In IgM and IgE antibody isotopes, the Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains CH2 and CH3 and the hinge between CH1 and CH2 domains. Although the definition of the boundaries of the Fc region of an immunoglobulin heavy chain might vary, as defined herein, the human IgG heavy chain Fc region is defined to stretch from an amino acid residue D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxy-terminus of the heavy chain, wherein the numbering is according to the EU index as in Kabat (Kabat, et al., 1991). The CH2 domain of a human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is positioned on C-terminal side of a CH2 domain in an Fc region, i.e., it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of an IgG. As used herein, the Fc region can be a native sequence Fc, including any allotypic variant, or a variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to this region in isolation or in the context of an Fc-comprising protein polypeptide such as a “binding protein comprising an Fc region,” also referred to as an “Fe fusion protein” (e.g., an antibody or immunoadhesin).

A “native sequence Fc region” or “native sequence Fc” has an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgG1 Fch region; native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof. Native sequence Fc include the various allotypes of Fcs. (See, e.g., Jefferis et al. (2009) mAbs 1:1).

The term “epitope” or “antigenic determinant” refers to a site on an antigen (e.g., hNKG2A protein) to which an immunoglobulin or antibody specifically binds, e.g., as defined by the specific method used to identify it. Epitopes can be formed both from (1) contiguous amino acids (usually a linear epitope) or (2) non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids in a unique spatial conformation.

The term “epitope mapping” refers to the process of identification of the molecular determinants for antibody-antigen recognition. Methods for determining what epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from the protein (e.g., from NKG2A) are tested for reactivity with a given antibody (e.g., anti-NKG2A antibody). Methods for determining spatial conformation of epitopes include techniques known in the art and those described herein, for example, x-ray crystallography; antigen mutational analysis, two-dimensional nuclear magnetic resonance; yeast display; and hydrogen/deuterium exchange-mass spectrometry (HDX-MS) (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

The term “binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether antibodies bind to the “same epitope on NKG2A” with the antibodies described herein include, for example, epitope mapping methods, such as x-ray analyses of crystals of antigen:antibody complexes, which provides atomic resolution of the epitope, HDX-MS, and fast photochemical oxidation of proteins (FPOP). Other methods monitor the binding of the antibody to antigen fragments (e.g. proteolytic fragments) or to mutated variations of the antigen, where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component, such as alanine scanning mutagenesis (Cunningham & Wells (1985) Science 244:1081) or yeast display of mutant target sequence variants. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies having the same VH and VL or the same CDR1, CDR2, and CDR3 sequences are expected to bind to the same epitope.

Antibodies that “compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, may be determined using known binding competition experiments, e.g., Biacore® surface plasmon resonance (SPR) analysis. In certain embodiments, an antibody competes with, and inhibits binding of another antibody to a target by at least 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition may be different depending on which antibody is the “blocking antibody” (i.e., the cold antibody that is incubated first with the target). Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb. Protoc. 2006; doi:10.1101/pdb.prot4277 or in Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA 1999. Two antibodies “cross-compete” if antibodies block each other both ways by at least 50%, i.e., regardless of whether one or the other antibody is contacted first with the antigen in the competition experiment.

Competitive binding assays for determining whether two antibodies compete or cross-compete for binding include competition for binding to T cells expressing NKG2A, e.g., by flow cytometry. Other methods include: SPR (e.g., Biacore®); solid phase direct or indirect radioimmunoassay (RIA); solid phase direct or indirect enzyme immunoassay (EIA); sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assay; solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, the terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen. In some embodiments, the antibody: (1) binds with an equilibrium dissociation constant (K_(D)) of approximately less than 10⁻⁶ M, such as approximately less than 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower when determined by, e.g., SPR technology in a Biacore® SPR instrument using the predetermined antigen, e.g., recombinant human NKG2A as the analyte and the antibody as the ligand, or Scatchard analysis of binding of the antibody to antigen positive cells; and (2) binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. Accordingly, an antibody that “specifically binds to human NKG2A” refers to an antibody that binds to soluble or cell bound human NKG2A with a K_(D) of 10⁻⁶ M or less, such as approximately less than 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower. An antibody that “cross-reacts with cynomolgus NKG2A” refers to an antibody that binds to cynomolgus NKG2A with a K_(D) of 10⁻⁶ M or less, such as approximately less than 10⁻⁷, 10⁻⁸ M, 10⁻⁹M or 10⁻¹⁰ M or even lower.

The terms “k_(a)”, “k_(assoc)”, or “k_(on)” are used interchangeably herein to refer to the association rate constant of a particular antibody-antigen interaction. The terms “k_(d),” “k_(dis)”, or “k_(off)” are used interchangeably herein to refer to the dissociation rate constant of a particular antibody-antigen interaction. The term “K_(D)”, as used herein, refers to the equilibrium dissociation constant, which is obtained from the ratio of k_(d) to k_(a) (i.e., k_(d)/k_(a)) and is expressed as a molar concentration (M). K_(D) values for antibodies can be determined using methods well established in the art. Available methods for determining the K_(D) of an antibody include but are not limited to surface plasmon resonance (SPR), using, for example, a biosensor system such as a Biacore® system, and flow cytometry and Scatchard analysis.

The term “IC₅₀” means half maximal inhibitory concentration, and measures the potency of a substance, for example, an antibody, to inhibit a specific biological or biochemical response. In other words, IC₅₀ is used as a measure of potency; the smaller the IC₅₀, the more potent the substance is. In the context of an in vitro or in vivo assay using an antibody, or antigen binding fragment thereof, IC₅₀ refers to the concentration of the antibody, or antigen-binding fragment thereof, that reduces the maximal biological or biochemical response by 50%.

The term “EC₅₀” means half maximal effective concentration, and measures the potency of a substance, for example, an antibody, to induce a specific biological or biochemical response. As with IC₅₀, EC₅₀ is used as a measure of potency; the smaller the EC₅₀, the more potent the substance is. In the context of an in vitro or in vivo assay using an antibody or antigen binding fragment thereof, EC₅₀ refers to the concentration of the antibody or antigen-binding fragment thereof that induces a response that is 50% of the maximal biological or biochemical response.

“Receptor occupancy” or “occupancy of the receptor,” as used herein, refers to the amount of antibody (e.g., the anti-NKG2A antibodies described herein) that is bound to the immunostimulatory receptor (e.g., human NKG2A). “Percent (%) receptor occupancy” or “percent (%) occupancy of the receptor” can be calculated using the following formula: ([ΔMFI of Test]/[ΔMFI of Total])×100. Change in mean fluorescence unit (ΔMFI) is calculated by subtracting the MFI of background staining with an isotype control antibody from the MFI from the bound antibody. The total receptor level is determined by adding a saturating amount of antibody to determine the maximum expression and, therefore, MFI of the particular immunostimulatory receptor. An alternative means to calculate total receptor expression is to use an antibody against the same immunostimulatory receptor that does not compete with the antibody for which receptor occupancy is being calculated.

As used herein, the term “naturally-occurring” as applied to a substance is a substance that is present in nature that has not been intentionally modified by people. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by people in the laboratory is naturally-occurring.

A “polypeptide” refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in the protein may contain a modification such as, but not limited to, glycosylation, phosphorylation or a disulfide bond. A “protein” comprises one or more polypeptides.

The term “nucleic acid molecule,” as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule is single-stranded or double-stranded, and may be complementary DNA (cDNA).

The term “cDNA” or “complementary DNA” refers to a non-naturally occurring nucleic acid molecule that has been created or derived from mRNA, i.e., the non-coding regions have been removed.

As used herein, the term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. “Conservative amino acid substitutions” refers to substitutions of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, a predicted nonessential amino acid residue in an anti-NKG2A antibody is replaced with another amino acid residue from the same side chain family. Methods of identifying nucleotide and amino acid conservative substitutions that do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

For nucleic acids, the term “substantial homology” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, at least about 90% to 95%, or at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions to the complement of the nucleic acid strand.

For polypeptides, the term “substantial homology” indicates that two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80% of the amino acids, at least about 90% to 95%, or at least about 98% to 99.5% of the amino acids.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent homology=(number of identical positions)/(total number of positions)×100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

The percent identity between two nucleotide sequences can be determined, e.g., using the GAP program in the GCG software package, using a nwsgapdna.cmp matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences described herein can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the) (BLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g.,)(BLAST and NBLAST) can be used. (See, e.g., National Center for Biotechnology Information (NCBI), available at https://www.ncbi.nlm.nih.gov/).

The nucleic acids may be present in whole cells, e.g., a host cell, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., the other parts of the chromosome) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. (See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)).

The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). Expression vectors useful in recombinant DNA techniques include plasmids. As used herein, “plasmid” and “vector” may be used interchangeably, as the plasmid is the most commonly used form of vector. However, also included are other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term “host cell” or “recombinant host cell”, which are used interchangeably, refers to a cell that comprises a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

An “immune response” is as understood in the art, and generally refers to a biological response in a vertebrate against foreign agents or abnormal, e.g., cancerous cells, where the response protects the vertebrate against these agents and diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal cells or tissues, including, for example, human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell or a T helper (Th) cell, such as a CD4+ or CD8+ T cell, or the inhibition or depletion of a Treg cell.

“Effector T” (“Teff”) cells are T cells (e.g., CD4+ and CD8+ T cells) with cytolytic activities as well as T helper (Th) cells. Th cells secrete cytokines and activate and direct other immune cells, but does not include regulatory T cells (Treg cells).

T regulatory (“Treg”) cells are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Memory B cells are a B cell sub-type that are formed within germinal centers following primary infection and are important in generating an accelerated and more robust antibody-mediated immune response in the case of re-infection (also known as a secondary immune response).

“Natural killer” (NK) cells are important mediators of the immune response against pathogens and tumors, and are part of the innate immune system. NK cells also have a role in regulating the adaptive immune response, and have been shown in different contexts to stimulate or inhibit T cell responses. NK cells provide rapid responses to viral-infected cells and respond to tumor formation.

As used herein, the term “T cell-mediated response” refers to a response mediated by T cells, e.g., effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells). T cell mediated responses include, for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refers to an immune response induced by cytotoxic T cells. CTL responses are mediated by, for example, CD8+ T cells.

An “immunomodulator” or “immunoregulator” refers to an agent, e.g., a component of a signaling pathway that may be involved in modulating, regulating, or modifying an immune response. “Modulating,” “regulating,” or “modifying” an immune response refers to any alteration in a cell of the immune system or in the activity of such cell (e.g., an effector T cell, such as a Th1 cell). Such modulation includes stimulation or suppression of the immune system, which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, and/or any other changes that can occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in a tumor microenvironment. In some embodiments, the immunomodulator is located on the surface of a T cell. An “immunomodulatory target” or “immunoregulatory target” is an immunomodulator that is targeted for binding by, and whose activity is altered by the binding of, a substance, agent, moiety, compound or molecule. Immunomodulatory targets include, for example, receptors on the surface of a cell (“immunomodulatory receptors”) and receptor ligands (“immunomodulatory ligands”).

“Immunotherapy” refers to the treatment of a subject, such as a human subject, afflicted with or at risk of contracting or suffering a recurrence of a disease by a method comprising inducing, enhancing, suppressing, or otherwise modifying an immune response.

“Immunostimulating therapy” or “immunostimulatory therapy” refers to a therapy that results in increasing (inducing or enhancing) an immune response in a subject for, e.g., treating cancer.

“Potentiating an endogenous immune response” means increasing the effectiveness or potency of an existing immune response in a subject, such as a human subject. This increase in effectiveness and potency may be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

As used herein, the term “linked” refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage also can be genetic (i.e., recombinantly fused). Such linkages can be achieved using a wide variety of art recognized techniques, such as chemical conjugation and recombinant protein production.

As used herein, “administering” refers to the physical introduction of a composition comprising a therapeutic agent, e.g., an anti-NKG2A antibody, to a subject, using any of the various methods and delivery systems known to those skilled in the art. “Administering” includes, for example, administration to a human patient by another, such as, for example, one or more healthcare providers, and self-administration by the human patient. Various routes of administration for antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example, by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, such as by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, an antibody described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

As used herein, “adjunctive” or “combined” administration (coadministration) includes simultaneous administration of the compounds in the same or different dosage form, or separate administration of the compounds (e.g., sequential administration). Thus, a first antibody, e.g., the anti-NKG2A antibody, and a second, third, or more antibodies can be simultaneously administered in a single formulation. Alternatively, the first and second (or more) antibodies can be formulated for separate administration and are administered concurrently or sequentially. “Combination” therapy, as used herein, means administration of two or more therapeutic agents in a coordinated fashion, and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g. administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time. (See, e.g., Kohrt et al. (2011) Blood 117:2423).

For example, the anti-NKG2A antibody can be administered first followed by (e.g., immediately followed by) the administration of a second antibody, or vice versa. In one embodiment, the anti-NKG2A antibody is administered prior to administration of the second antibody. In another embodiment, the anti-NKG2A antibody is administered, for example, within about 30 minutes of the second antibody. Such concurrent or sequential administration preferably results in both antibodies being simultaneously present in treated patients.

As used herein, the terms “inhibits” or “blocks” are used interchangeably and encompass both partial and complete inhibition/blocking. In some embodiments, the anti-NKG2A antibody described herein inhibits binding of NKG2A to HLA-E by at least about 50%, for example, about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, determined, e.g., as further described herein. In some embodiments, the anti-NKG2A antibody inhibits binding of NKG2A to HLA-E by no more than 50%, for example, by about 40%, 30%, 20%, 10%, 5% or 1%, determined, e.g., as further described herein.

As used herein, “cancer” refers a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell growth or division may result in the formation of malignant tumors or cells that invade neighboring tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream.

The terms “treat,” “treating,” and “treatment,” as used herein, refer to any type of intervention or process performed on, or administering an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. In contrast, “prophylaxis” or “prevention” refers to administration to a subject who does not have a disease to prevent the disease from occurring. As used herein, “treat,” “treating,” and “treatment” does not encompass prophylaxis or prevention.

The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A “therapeutically effective amount” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. A “prophylactically effective amount” or a “prophylactically effective dosage” of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of disease, prevents the development or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or of a prophylactic agent to prevent the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

The administration of effective amounts of the anti-NKG2A antibody alone, or anti-NKG2A antibody combined with, for example, an anti-PD-1 antibody, combined with an anti-PD-L1 antibody, or combined with an anti-CTLA-4 antibody, according to any of the methods provided herein, can result in at least one therapeutic effect, including, for example, reduced tumor growth or size, reduced number of metastatic lesions appearing over time, complete remission, partial remission, or stable disease. For example, the methods of treatment produce a comparable clinical benefit rate (CBR=complete remission (CR)+ partial remission (PR)+stable disease (SD) lasting ≥6 months) better than that achieved without administration of the anti-NKG2A antibody, or than that achieved with administration of any one of the combined antibodies alone, e.g., the improvement of clinical benefit rate is about 20%, 30%, 40%, 50%, 60%, 70%, 80% or more.

By way of example, an anti-cancer agent is a drug that slows cancer progression or promotes cancer regression in a subject, including a human subject. In some embodiments, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer. “Promoting cancer regression” means that administering an effective amount of the drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, necrosis of the tumor, a decrease in severity of at least one disease symptom, an increase in frequency and duration of disease symptom-free periods, a prevention of impairment or disability due to the disease affliction, or otherwise amelioration of disease symptoms in the patient. “Pharmacological effectiveness,” “effectiveness,” or “efficacy” refers to the ability of the drug to promote cancer regression in the patient. “Physiological safety” refers to an acceptably low level of toxicity or other adverse physiological effects at the cellular, organ and/or organism level (adverse effects) resulting from administration of the drug.

By way of example, for the treatment of tumors, a therapeutically effective amount or dosage of the drug inhibits tumor cell growth by at least about 20%, by at least about 30% by at least about 40%, by at least about 50%, by at least about 60%, by at least above 70%, by at least about 80% relative to untreated subjects, or by at least about 90%. In some embodiments, a therapeutically effective amount or dosage of the drug completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by 100%. The ability of a compound, including an antibody, to inhibit tumor growth can be evaluated using the assays described herein. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth; such inhibition can be measured in vitro by assays known to person of ordinary skill in the art. In some embodiments, inhibition of tumor growth may not be immediate after treatment, and may only occur after a period of time or after repeated administration. In other embodiments described herein, tumor regression is observed and continues for at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days, or longer.

As used herein, the terms “fixed dose”, “flat dose” and “flat-fixed dose” are used interchangeably and refer to a dose that is administered to a patient without regard for the weight or body surface area of the patient. The fixed or flat dose is therefore not provided as a mg/kg dose, but rather as an absolute amount of the therapeutic agent.

As used herein, the term “weight based” dose or dosing means that a dose administered to a patient is calculated based on the patient's weight. For example, when a 60 kg patient requires 3 mg/kg of an anti-NKG2A antibody, one can calculate and use the appropriate amount of the anti-NKG2A antibody (i.e., 180 mg) for administration.

The term “patient” includes human and other mammalian subjects that receive either therapeutic or prophylactic treatment.

The term “subject” includes any human or non-human animal. For example, the methods and compositions herein disclosed can be used to treat a subject having cancer. A non-human animal includes all vertebrates, e.g., mammals and non-mammals, including non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc. In one embodiment, the subject is a human subject.

As used herein, the term “a” or “an” entity refers to one or more of that entity unless otherwise specified; for example, “a nucleotide sequence” is understood to represent one or more nucleotide sequences. As such, the terms “a” or “an”, “one or more,” and “at least one” can be used interchangeably herein.

As used herein, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” includes “A and B,” “A or B,” “A” alone, and “B” alone. Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” encompasses each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided.

Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form unless otherwise indicated. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5′ to 3′ orientation. Amino acid sequences are written left to right in amino to carboxy orientation.

As used herein, the term “about” or “approximately” means roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).

The headings provided herein are not limitations of the various aspects of the disclosure, and should be read by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. Various aspects described herein are described in further detail in the following subsections.

I. Anti-NKG2A Antibodies

The present disclosure describes, in some embodiments, anti-NKG2A antibodies, such as fully human, humanized, and chimeric antibodies, with desirable functions or properties. For example, the antibodies specifically bind human NKG2A protein with high affinity. In certain embodiments, the antibodies are antagonistic antibodies that block or reverse NKG2A-mediated inhibition on immune cells such as T cells and NK cells. In some embodiments, the anti-human NKG2A (anti-huNKG2A) antibodies have desirable properties for use as therapeutic agents in treating diseases such as cancers or infections.

Specific anti-NKG2A antibodies described herein are antibodies having the CDR and/or variable region sequences of antibodies 13F3.A4, NKG2A.6, NKG2A.7, NKG2A.8, NKG2A.9, NKG2A.11, isolated and structurally characterized as described herein, as well as antibodies having at least 80% identity (for example, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to the amino acid sequences of the anti-NKG2A antibodies described herein. In some embodiments, the anti-NKG2A antibodies described herein have at least 80% identity (for example, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to the variable region or CDR sequences of the anti-NKG2A antibodies described herein.

In some aspects, the antibodies of the invention are characterized by particular functional features or properties. For example, the antibodies specifically bind to human NKG2A with high affinity. In some embodiments, the anti-NKG2A antibodies inhibits the binding of NKG2A to its ligand HLA-E, which restores NK and T cell responses against tumors that express HLA-E. In other words, the anti-NKG2A antibodies described herein stimulate T cell and NK cell anti-tumor responses by inhibiting or blocking the interaction between NKG2A protein and its ligand HLA-E.

In some embodiments, the Anti-NKG2A antibodies described herein exhibit one or more of the following properties:

-   -   (1) Binds specifically to human NKG2A protein;     -   (2) Blocks or reduces binding and/or interaction of an NKG2A         ligand (e.g., HLA-E in humans) to human NKG2A protein; (In other         embodiments, the anti-NKG2A antibodies described herein blocks         or reduces binding and/or interaction of an NKG2A ligand to         non-human NKG2A protein);     -   (3) Reverses NKG2A-mediated inhibitory signaling;     -   (4) Does not bind, or binds with low affinity to, human NKG2C         protein;     -   (5) Binds with high affinity to human and cynomolgus monkey         NKG2A     -   (6) Does not bind, or shows low affinity to, mouse or rat NKG2A;     -   (7) Does not interfere with activating signal from HLA-E binding         to NKG2C protein;     -   (8) Has reduced binding to human Fc gamma receptor (FcγR);     -   (9) Induces and/or enhances an anti-tumor immune response;     -   (10) Enhances the functional activity of T cells; (In some         embodiments, increases cytototoxic T cell function as measured         by, for example, lysis of HLA-E-expressing tumor cells);     -   (11) Enhances the functional activity of natural killer (NK)         cells by, for example, inducing NK cell activation;     -   (12) Increases cytokine, for example, IFNγ production; and/or     -   (13) Specifically binds to an epitope located within         discontinuous regions comprising the following amino acid         residues as determined by HDX-MS and/or FPOP epitope mapping:         -   Region 1: ¹⁵⁵LSIDNEEEMKF¹⁶⁵ (amino acid residues 155 to 165             of SEQ ID NO: 2;         -   Region 2: ¹⁷¹PSSWIGVFRNSSHHPW¹⁸⁶ (amino acid residues 171 to             186 of SEQ ID NO: 2;         -   Region 3: ¹⁹²LAFKHEIKDSDN²⁰³ (amino acid residues 192 to 203             of SEQ ID NO: 2;         -   Region 4: L (amino acid residue 206 of SEQ ID NO: 2); and         -   Region 5: ²¹²QVNRLKSAQCGSSIIYHC²²⁹ (amino acid residues 212             to 229 of SEQ ID NO: 2).     -   (14) Specifically binds to an epitope located within         discontinuous regions comprising the following amino acid         residues as determined by HDX-MS: Region 1: ¹⁵⁵LSIDNEEEMKF¹⁶⁵         (amino acid residues 155 to 165 of SEQ ID NO: 2)     -   Region 2: ¹⁷¹PSSWIGVFRNSSHHPW¹⁸⁶ (amino acid residues 171 to 186         of SEQ ID NO: 2);     -   Region 3: ¹⁹²LAFKHEIKDSDN²⁰³ (amino acid residues 192 to 203 of         SEQ ID NO: 2); and     -   Region 5: ²¹²QVNRLKSAQCGSSIIYHC²²⁹ (amino acid residues 212 to         229 of SEQ ID NO: 2).

In some embodiments, the anti-NKG2A antibodies described herein bind with high affinity to human and cynomolgus monkey NKG2A, and does not bind to, or binds with low affinity to non-primate NKG2A, such as mouse or rat NKG2A. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit one or more of the following properties:

(a) has an EC₅₀ value of about 0.6 nM or lower for binding to human NKG2A protein as measured by cell binding assay;

(b) has an EC₅₀ value of about 9.0 nM or higher for binding to human NKG2C protein as measured by cell binding assay;

(c) has an IC₅₀ value of about 1.0 nM or lower for reducing the binding and/or interaction of HLA-E to human NKG2A protein as measured by cell blocking assay;

(d) binds to human NKG2A protein with a K_(D) of about 0.4 nM or lower as measured by Scatchard analysis;

(e) binds to human NKG2A protein with a K_(D) of about 61 nM or lower as measured by surface plasmon resonance;

(f) binds to cynomolgus NKG2A protein with a K_(D) of about 1.0 nM or lower as measured by Scatchard analysis;

(g) is internalized upon binding to NKG2A-expressing cells;

(h) increases interferon-gamma (IFNγ) production; and/or

(i) wherein the half-life of the anti-NKG2A antibody:NKG2A protein complex is about 40 seconds or longer.

In some embodiments, the anti-NKG2A antibodies described herein block binding and/or interaction of an NKG2A ligand (HLA-E in humans) to human NKG2A protein. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit one or more of the following properties:

-   -   a) Anti-NKG2A antibody blocks HLA-E pentamer from binding to         cells expressing human NKG2A with an IC₅₀ of about 0.30 nM for         NKL and an IC₅₀ of about 1.0 nM for CHO-hNKG2A cells;     -   b) Anti-NKG2A antibody complexed with either HNKG2A-CD94-mFC or         cynomolgus NKG2A-CD94-mFc protein blocks human HLA-E binding.

In some embodiments, the anti-NKG2A antibodies described herein is specific to human NKG2A. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit one or more of the following properties:

-   -   a) Has an EC₅₀ value for the anti-NKG2A antibody binding to         human NKG2A that is about 15-fold less than a second EC₅₀ value         for the anti-NKG2A antibody binding to human NKG2C protein. In         some embodiments, the EC₅₀ value for the anti-NKG2A antibody         binding to human NKG2A was about 0.6 nM, while the EC₅₀ value         for the anti-NKG2A antibody binding to human NKG2C was about 9.0         nM.     -   b) No specific binding of the anti-NKG2A antibody to human NKG2C         based on SPR analysis; and/or     -   c) Does not block human NKG2C and HLA-E interaction as measured         by flow cytometry.

In some embodiments, the anti-NKG2A has an inert Fc (for example, the antibody is IgG1 isotype) to reduce or prevent FcγR binding. Although not bound by any theory, since NKG2A is an inhibitory receptor expressed on CD8+ T and NK cells, reducing agonism or depletion of NKG2A+CD8+ T or NK cells is beneficial for anti-tumor function. Thus, blocking NKG2A and HLA-E interaction can be done with an anti-NKG2A antibody that does not interact with human FcγRs.

In some embodiments, the anti-NKG2A described herein enhances the anti-tumor functional activity of T cells. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit one or more of the following properties:

-   -   a) Reverses the inhibition of NK-κB signaling in a         NKG2A-expressing Jurkat T cell line stimulated by         CHO/scOKT3/HLA-E, with an EC₅₀ value of about 0.2 nM or lower.     -   b) Induces interferon-gamma (IFN-γ) in NKG2A⁺ CD8 T isolated         from healthy donor PBMC co-cultured with CHO/scOKT3/HLA-E/PD-L1.     -   c) Induces IFN-γ in NKG2A⁺ CD8 T cells isolated from human         tumors co-cultured with CHO/scOKT3/HLA-E/PD-L1.

In some embodiments, the anti-NKG2A antibodies described herein enhances the anti-tumor functional activity of NK cells. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit one or more of the following properties:

-   -   a) Increases IFN-γ production in NKL cells co-cultured with         CHO/MICA/HLA-E.     -   b) Induces a dose-dependent increase in NK cell degranulation         and lysis of HLA-E-expressing tumor cells.

In some embodiments, the anti-NKG2A antibodies described herein is internalized after binding to NKG2A-expressing cells. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit internationalization with an EC₅₀ of about 0.5 nM lower or lower. In some embodiments, the anti-NKG2A antibodies exhibit dose-dependent internationalization with an EC₅₀ of about 0.5 nM lower or lower.

In some embodiments, the anti-NKG2A antibodies of the invention lack sequence liabilities that reduce the chemical stability of the antibodies. The anti-NKG2A antibodies of the invention have various important uses, e.g., for treatment and/or diagnosis of cancer and other disorders associated with NKG2A expression and/or activity.

In some embodiments, the anti-NKG2A antibodies disclosed herein by amino acid sequence bind to specific epitopes on human NKG2A, as described in Example 4.

Binding to human NKG2A can be assessed using one or more techniques well established in the art. For example, in some embodiments, the antibody is tested by a flow cytometry assay in which the antibody is reacted with a cell line that expresses human NKG2A, such as CHO cells that have been transfected to express human NKG2A on their cell surface. Additionally or alternatively, the binding of the antibody, including the binding kinetics (e.g., K_(D) value) can be tested in Biacore binding assays. Still other suitable binding assays include ELISA assays using, for example, a recombinant human NKG2A protein.

In some embodiments, anti-NKG2A antibodies or antigen-binding fragments thereof described herein bind to human NKG2A protein with high affinity, for example, with nanomolar affinity, including with a K_(D) of 1×10⁻⁶ M or lower, 1×10⁻⁷ M or lower, 1×10⁻⁸M or lower, 1×10⁻⁹M or lower, or 10⁻¹⁰M or lower.

Some embodiments of the present invention relates to an anti-NKG2A monoclonal antibody or antigen-binding portion thereof that specifically binds to an epitope located within discontinuous regions comprising the following amino acid residues as determined by HDX-MS and/or FPOP epitope mapping:

-   -   Region 1: ¹⁵⁵LSIDNEEEMKF¹⁶⁵ (amino acid residues 155 to 165 of         SEQ ID NO: 2 (native hNKG2A amino acid sequence);     -   Region 2: ¹⁷¹PSSWIGVFRNSSHHPW¹⁸⁶ (amino acid residues 171 to 186         of SEQ ID NO: 2);     -   Region 3: ¹⁹²LAFKHEIKDSDN²⁰³ (amino acid residues 192 to 203 of         SEQ ID NO: 2);     -   Region 4: L (amino acid residue 206 of SEQ ID NO: 2); and     -   Region 5: ²¹²QVNRLKSAQCGSSIIYHC²²⁹ (amino acid residues 212 to         229 of SEQ ID NO: 2).

In some embodiments, the invention is directed to an anti-NKG2A monoclonal antibody or antigen-binding portion thereof that specifically binds to an epitope located within discontinuous regions comprising the following amino acid residues as determined by HDX-MS:

-   -   Region 1: ¹⁵⁵LSIDNEEEMKF¹⁶⁵ (amino acid residues 155 to 165 of         SEQ ID NO: 2);     -   Region 2: ¹⁷¹PSSWIGVFRNSSHHPW¹⁸⁶ (amino acid residues 171 to 186         of SEQ ID NO: 2);     -   Region 3: ¹⁹²LAFKHEIKDSDN²⁰³ (amino acid residues 192 to 203 of         SEQ ID NO: 2); and     -   Region 5: ²¹²QVNRLKSAQCGSSIIYHC²²⁹ (amino acid residues 212 to         229 of SEQ ID NO: 2.

In some embodiments, the anti-NKG2A antibodies described herein enhance NK cell function by blocking NKG2A/HLA-E-mediated inhibition. In another embodiment, the anti-NKG2A antibody binds to human NKG2A protein and stimulates an anti-tumor immune response, e.g., an antigen-specific T cell and/or NK cell response. The ability of the anti-NKG2A antibody to stimulate an immune response can be tested by measuring tumor growth, such as in an in vivo tumor graft model, as described in the Examples herein. In other embodiments, the anti-NKG2A antibodies or antigen binding portions thereof increase cytokine production (e.g., interferon-gamma (IFN-γ) in NKG2A-expressing T cells and/or increase T cell proliferation, including effector T cells and cytotoxic T cells (also known as CD8⁺ T cells).

In another embodiment, the anti-NKG2A antibody, or antigen-binding fragment thereof, binds to human NKG2A and exhibits at least one of the following properties:

-   -   a) binds to one or more of the following residues as determined         by HDX-MS and/or FPOP epitope mapping:     -   Region 1: ¹⁵⁵LSIDNEEEMKF¹⁶⁵ (amino acid residues 155 to 165 of         SEQ ID NO: 2 (native hNKG2A amino acid sequence);     -   Region 2: ¹⁷¹PSSWIGVFRNSSHHPW¹⁸⁶ (amino acid residues 171 to 186         of SEQ ID NO: 2);     -   Region 3: ¹⁹²LAFKHEIKDSDN²⁰³ (amino acid residues 192 to 203 of         SEQ ID NO: 2);     -   Region 4: L (amino acid residue 206 of SEQ ID NO: 2); and     -   Region 5: ²¹²QVNRLKSAQCGSSIIYHC²²⁹ (amino acid residues 212 to         229 of SEQ ID NO: 2);     -   b) binds to the same epitope on human NKG2A as NKG2A.11 and         13F3.A4 antibodies;     -   c) competes for binding to human NKG2A with NKG2A.11 and 13F3.A4         antibodies;     -   d) binds to human NK cell with an EC₅₀ of about 0.4 nM as         measured by Biacore;     -   e) blocks binding of human NK cells to HLA-E with an IC₅₀ of         about 0.3 nM as measured by Biacore;     -   f) binds to cynomolgus monkey NKG2A-expressing CHO cells with an         EC₅₀ of about 1 nM or lower;     -   g) has low binding to human NKG2C with an EC₅₀ of about 9.0 nM         or higher (in other words, the anti-NKG2A antibody does not         block the interaction of human NKG2C to HLA-E); and/or     -   h) enhances CD8+ T cell and NK cell anti-tumor responses. For         example:         -   i. increases IFN-γ production in a primary             T:CHO-OKT3-HLA-E-PDL1 assay;         -   ii. increases IFN-γ production in T cell tumor-infiltrating             lymphocytes (TILs):CHO-OKT3-HLA-E-PDL1 assay; and/or         -   iii. increases cytoxocity and IFN-γ production in primary NK             cell assays.

In some embodiments, the anti-NKG2A antibodies of the invention include humanized and fully human monoclonal antibodies. In other embodiments, the antibodies are, for example, chimeric monoclonal antibodies.

a. Anti NKG2A Monoclonal Antibodies

In some embodiments, the antibodies of the invention are monoclonal antibodies 13F3.A4, NKG2A.9, and NKG2A.11, which are isolated and structurally characterized as described in the following Examples. The VH and the VL amino acid sequences are set forth in the Sequence Table and Sequence Listing.

Given that each of these antibodies can bind to human NKG2A, the VH and VL sequences can be “mixed and matched” to create other anti-hNKG2A binding molecules of the invention. In some embodiments, when VH and VL chains are mixed and matched, a VH sequence from a particular VH/VL pairing is replaced with a structurally similar VH sequence. Likewise, in some embodiments, a VL sequence from a particular VH/VL pairing is replaced with a structurally similar VL sequence. Accordingly, in one aspect, this disclosure provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that binds to human NKG2A protein, wherein the light and heavy chain variable regions comprise:

(a) the amino acid sequences of SEQ ID NOs: 9 and 8, respectively;

(b) the amino acid sequences of SEQ ID NOs: 164 and 8, respectively; or

(c) the amino acid sequences of SEQ ID NOs: 169 and 167, respectively.

In another aspect, this disclosure provides antibodies that comprise the heavy chain and light chain CDR1s, CDR2s and CDR3s of NKG2A.9, NKG2A.11, and 13F3A.4 antibodies. Accordingly, in one aspect, this disclosure provides an isolated monoclonal antibody, or antigen binding fragment thereof, that binds to human NKG2A protein, wherein the antibody comprises:

(a) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 10, 11, 12, respectively, and a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively;

(b) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 154, 14, and 15, respectively; or

(c) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 region comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 155, 14, and 15, respectively.

It is well known in the art that the CDR3 domain, independently from the CDR1 and/or CDR2 domain(s), alone can determine the binding specificity of an antibody for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. (See, e.g., Klimka et al., British J. of Cancer 83(2):252-260 (2000). Accordingly, the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domains from an antibody derived from a human or non-human animal, wherein the monoclonal antibody is capable of specifically binding to human NKG2A. In certain aspects, the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human antibody, wherein the monoclonal antibody is capable of specifically binding to human NKG2A. Within some embodiments, such inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human antibody (a) are capable of competing for binding with; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the corresponding parental non-human antibody.

In other aspects, the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a human antibody, such as, e.g., a human antibody obtained from a non-human animal, wherein the human antibody is capable of specifically binding to human NKG2A. In other aspects, the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a first human antibody, such as, for example, a human antibody obtained from a non-human animal, wherein the first human antibody is capable of specifically binding to human NKG2A, and wherein the CDR3 domain from the first human antibody replaces a CDR3 domain in a human antibody that lacks binding specificity for NKG2A to generate a second human antibody that is capable of specifically binding to human NKG2A. In some embodiments, such inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from the first human antibody (a) are capable of competing for binding with; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the corresponding parental non-human antibody.

In some embodiments, the present invention provides anti-hNKG2A antibodies with inert Fc human IgG1.3 as the isotype. In some embodiments, such anti-hNKG2A antibodies with an inert Fc exhibit superior efficacy in treating cancer compared to other isotypes.

B. Antibodies with Conservative Modifications

In certain embodiments, an anti-NKG2A antibody of the invention comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences comprise specified amino acid sequences based on the antibodies described herein (e.g., the 13F3.A4, NKG2A.9, and NKG2A.11 antibodies), or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-hNKG2A antibodies of the invention. It is understood in the art that certain conservative sequence modifications can be made that do not remove antigen binding. (See, e.g., Brummell et al. (1993) Biochem 32:1180-8). Accordingly, this disclosure provides an isolated monoclonal antibody, or antigen binding fragment thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:

(a) the heavy chain variable region comprising a CDR3 sequence comprising an amino acid sequence set forth in SEQ ID NO: 12, or conservative modifications thereof; and

(b) the antibody, or antigen binding portion thereof, specifically binds human NKG2A. In additional embodiments, the antibody has one or more of the functional properties described herein, such as high affinity binding to human NKG2A, and/or the ability to block the NKG2A/HLA-E interaction.

In some embodiments, the heavy chain variable region comprising a CDR2 sequence comprises an amino acid sequence set forth in SEQ ID NO: 11, or conservative modifications thereof; and the light chain variable region comprising a CDR2 sequence comprising an amino acid sequence set forth in SEQ ID NO: 14, or conservative modifications thereof. In another embodiment, the heavy chain variable region comprises a CDR1 sequence comprising an amino acid sequence set forth in SEQ ID NO: 10, or conservative modifications thereof; and the light chain variable region comprising a CDR1 sequence comprising an amino acid sequence set forth in SEQ ID NOs: 13, 154, or 155, or conservative modifications thereof.

In various embodiments, the anti-NKG2A antibody is, for example, human antibodies, humanized antibodies, or chimeric antibodies.

c. Antibodies that Bind to the Same Epitope as Anti-hNKG2A Antibodies

In another embodiment, this disclosure provides antibodies that bind to the same epitope on human NKG2A protein as any of the anti-hNKG2A monoclonal antibodies of the invention (i.e., antibodies that have the ability to cross-compete for binding to human NKG2A protein with any of the monoclonal antibodies of the invention). In some embodiments, the reference antibody for cross-competition studies are the monoclonal antibodies NKG2A.9, NKG2A.11, and 13F3.A4 in standard human NKG2A binding assays. For example, standard ELISA assays can be used in which a recombinant human NKG2A protein is immobilized on the plate, one of the antibodies is fluorescently labeled, and the ability of non-labeled antibodies to compete off the binding of the labeled antibody is evaluated. Additionally or alternatively, Biacore analysis can be used to assess the antibodies' ability to cross-compete. The ability of a test antibody to inhibit the binding of, for example, NKG2A.9, NKG2A.11, and/or 13F3.A4, to human NKG2A demonstrates that the test antibody can compete with NKG2A.9, NKG2A.11, and/or 13F3.A4 for binding to human NKG2A and thus binds to the same epitope on human NKG2A.9 as NKG2A.9, NKG2A.11, and/or 13F3.A4. In some embodiments, the antibody that binds to the same epitope on human NKG2A as NKG2A.9, NKG2A.11, and/or 13F3.A4 is a humanized or human monoclonal antibody.

As discussed further in Example 4, the binding of NKG2A.9 and 13F3.A4 has been mapped to specific residues. Accordingly, in one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that, when bound to human NKG2A protein, the antibody, or antigen-binding fragment thereof, specifically binds to the following amino acid residues as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS):

(e) (SEQ ID NO: 156) LSIDNEEMKF; (f) (SEQ ID NO: 157) PSSWIGVFRNSSHHPW; (g) (SEQ ID NO: 158) LAFKHEIKDSDN; and (h) (SEQ ID NO: 159) QVNRLKSAQQCGSSIIYHC,

-   -   wherein the monoclonal antibody blocks the binding of an NKG2A         ligand (e.g., HLA-E in humans) to human NKG2A protein.         In another embodiment, the invention provides an isolated         monoclonal antibody, or antigen-binding fragment thereof, that,         when bound to human NKG2A, the monoclonal antibody specifically         binds to the following amino acid residues as determined by         HDX-MS and/or fast photochemical oxidation of proteins (FPOP)         epitope mapping:

(f) (SEQ ID NO: 156) LSIDNEEMKF; (g) (SEQ ID NO: 157) PSSWIGVFRNSSHHPW; (h) (SEQ ID NO: 158) LAFKHEIKDSDN; (i) L; and (j) (SEQ ID NO: 159) QVNRLKSAQQCGSSIIYHC, wherein the monoclonal antibody blocks the binding of an NKG2A ligand (e.g., HLA-E in humans) to human NKG2A protein.

Such humanized or human monoclonal antibodies can be prepared and isolated as described herein. For example, anti-hNKG2A antibodies that bind to the same or similar epitopes to the antibodies disclosed herein may be raised using immunization protocols, e.g., those described herein. The resulting antibodies can be screened for high affinity binding to human NKG2A. Selected antibodies can then be studied, e.g., in yeast display assay in which sequence variants of hNKG2A are presented on the surface of yeast cells, or by hydrogen-deuterium exchange experiments and/or FPOP, to determine the precise epitope bound by the antibody.

Epitope determinations may be made by any method known in the art. In some embodiments, anti-hNKG2A antibodies are considered to bind to the same epitope as an anti-anti-hNKG2A mAb disclosed herein if they make contact with one or more of the same residues within at least one region of hNKG2A; if they make contacts with a majority of the residues within at least one region of hNKG2A; if they make contacts with a majority of the residues within each region of hNKG2A; if they make contact with a majority of contacts along the entire length of hNKG2A; if they make contacts within all of the same distinct regions of hNKG2A; if they make contact with all of the residues at any one region on hNKG2A; or if they make contact with all of the same residues at all of the same regions. Epitope “regions” are clusters of residues along, but not necessarily directly adjacent within, the primary sequence.

Techniques for determining antibodies that bind to the “same epitope on hNKG2A” with the antibodies described herein include x-ray analyses of crystals of antigen:antibody complexes, which provides atomic resolution of the epitope. Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to an amino acid modification within the antigen sequence indicates the epitope component. Methods may also rely on the ability of an antibody of interest to affinity isolate specific short peptides (either in native three dimensional form or in denatured form) from combinatorial phage display peptide libraries or from a protease digest of the target protein. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed that have been shown to map conformational discontinuous epitopes.

The epitope or region comprising the epitope can also be identified by screening for binding to a series of overlapping peptides spanning NKG2A. Alternatively, the method of Jespers et al. (1994) Biotechnology 12:899 may be used to guide the selection of antibodies having the same epitope and therefore similar properties to the anti-NKG2A antibodies described herein. Using phage display, first, the heavy chain of the anti-NKG2A antibody is paired with a repertoire of (e.g., human) light chains to select an NKG2A-binding antibody, and then the new light chain is paired with a repertoire of (e.g., human) heavy chains to select a (e.g., human) NKG2A-binding antibody having the same epitope or epitope region as an anti-NKG2A antibody described herein. Alternatively, variants of an antibody described herein can be obtained by mutagenesis of cDNA sequences encoding the heavy and light chains of the antibody.

Alanine scanning mutagenesis, as described by Cunningham & Wells, Science 244: 1081 (1989), or some other form of point mutagenesis of amino acid residues in NKG2A may also be used to determine the functional epitope for an anti-NKG2A antibody.

The epitope or epitope region (an “epitope region” is a region comprising the epitope or overlapping with the epitope) bound by a specific antibody may also be determined by assessing binding of the antibody to peptides comprising NKG2A fragments. A series of overlapping peptides encompassing the NKG2A sequence (e.g., human NKG2A) may be synthesized and screened for binding, e.g. in a direct ELISA, a competitive ELISA (where the peptide is assessed for its ability to prevent binding of an antibody to NKG2A bound to a well of a microtiter plate), or on a chip. Such peptide screening methods may not be capable of detecting some discontinuous functional epitopes, i.e., functional epitopes that involve amino acid residues that are not contiguous along the primary sequence of the NKG2A polypeptide chain.

An epitope may also be identified by MS-based protein footprinting, such as HDX-MS and Fast Photochemical Oxidation of Proteins (FPOP). HDX-MS may be conducted, e.g., as further described at Wei et al. (2014) Drug Discovery Today 19:95, the methods of which are specifically incorporated by reference herein. FPOP may be conducted as described, e.g., in Hambley & Gross (2005) J. American Soc. Mass Spectrometry 16:2057, the methods of which are specifically incorporated by reference herein.

The epitope bound by anti-NKG2A antibodies may also be determined by structural methods, such as X-ray crystal structure determination (e.g., WO2005/044853), molecular modeling and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the H-D exchange rates of labile amide hydrogens in NKG2A when free and when bound in a complex with an antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31:11335; Zinn-Justin et al. (1993) Biochemistry 32:6884).

Unless otherwise indicated, and with reference to the claims, the epitope bound by an antibody is the epitope as determined by HDX-MS methods.

Anti NKG2A Antibodies that Bind with High Affinity

In some embodiments, the anti-hNKG2A antibodies of the present invention bind to hNKG2A with high affinity, making them effective therapeutic agents. In various embodiments, anti-hNKG2A antibodies of the present invention bind to hNKG2A with a K_(D) of less than 10 nM, 5 nM, 2 nM, 1 nM, 300 pM or 100 pM. Standard assays to evaluate the binding ability of the antibodies toward hNKG2A include ELISAs, RIAs, Western blots, biolayer interferometry (BLI) and Biacore™ SPR analysis (see Example 10).

d Anti NKG2A Antibody Sequence Variants

Anti-NKG2A antibody sequence variants disclosed herein maintain the desirable functional properties disclosed herein. The CDR regions are delineated using the Kabat system (Kabat, et al., 1991) unless otherwise specified. In some embodiments, the present invention further provides human or humanized anti-hNKG2A antibodies comprising CDR sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the CDR sequences of the antibodies disclosed herein. The present invention also provides anti-anti-hNKG2A antibodies comprising heavy and/or light chain variable domain sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the heavy and/or light chain variable domain sequences of the antibodies disclosed herein, as well as anti-hNKG2A antibodies comprising full-length heavy and/or light chain sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the heavy and/or light chain sequences of the antibodies disclosed herein.

II. Engineered and Modified Antibodies

a. V_(H) and V_(L) Regions

Also provided are engineered and modified antibodies that can be prepared using an antibody having one or more of the V_(H) and/or V_(L) sequences disclosed herein as starting material to engineer a modified antibody, which modified antibody may have altered properties from the starting antibody. In some embodiments, an antibody as described herein was engineered by modifying one or more residues within one or both variable regions (i.e., V_(H) and/or V_(L)), for example, within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody as described herein was engineered by modifying residues within the constant region(s), for example, to alter the effector function(s) of the antibody.

In one embodiment, the variable region engineering includes CDR grafting. Such grafting is of particular use in humanizing non-human anti-NKG2A antibodies, e.g., anti-HNKG2A antibodies that compete for binding with the anti-hNKG2A antibodies disclosed herein and/or bind to the same epitope as the select anti-hNKG2A antibodies disclosed herein. Antibodies interact with target antigens predominantly through amino acid residues that are located in the heavy and light chain CDRs. The CDRs are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”). Expression vectors can be constructed such that they include CDR sequences from a specific reference (also called “parental”) antibody grafted onto framework sequences from a different antibody (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.). In some instances, the resulting recombinant antibody has properties that are similar to the parental antibody. The engineered antibody can then be further modified to acquire properties that are distinct from the parental antibody. In other instances, grafting the parental CDR sequences onto a framework abrogates certain characteristics of the parental antibody such that the recombinant antibody no longer has these characteristics. One exemplary characteristic is binding affinity with respect to an antigen. In such instances, it might be advantageous to modify the engineered antibody further to regain the desired characteristics of the parental antibody.

Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBase” human germline sequence database, as well as in Kabat, E. A., et al., 1991); Tomlinson, I. M., et al. (1992) “The Repertoire of Human Germline V_(H) Sequences Reveals about Fifty Groups of V_(H) Segments with Different Hypervariable Loops” J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory of Human Germ-line V_(H) Segments Reveals a Strong Bias in their Usage,” Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference.

In some embodiments, framework sequences for use in the antibodies described herein are those that are structurally similar to the framework sequences used by antibodies described herein. The V_(H) CDR1, 2, and 3 sequences and the V_(L) CDR1, 2, and 3 sequences can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain up to 20 amino acid substitutions, including conservative amino acid substitutions, as compared to the germline sequences. For example, it has been found that in certain instances, it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).

Engineered antibodies described herein include those in which modifications have been made to framework residues within V_(H) and/or V_(L), e.g., to improve the properties of the antibody, such as to decrease the immunogenicity of the antibody. For example, one approach is to “back-mutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be “back-mutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such “back-mutated” antibodies are also encompassed in this disclosure.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “de-immunization” and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

Another type of variable region modification is to mutate amino acid residues within the CDR regions to improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest. Preferably, conservative modifications are introduced. The mutations may be amino acid additions, deletions, or substitutions. In some embodiments, no more than one, two, three, four or five residues within a CDR region are altered.

Methionine residues in CDRs of antibodies can be oxidized, resulting in potential chemical degradation and consequent reduction in antibody potency. Accordingly, also provided herein are anti-NKG2A antibodies that have one or more methionine residues in the heavy and/or light chain CDRs replaced with amino acid residues that do not undergo oxidative degradation. Similarly, deamidation sites may be removed from anti-NKG2A antibodies, particularly in the CDRs. Also provided herein are antibodies in which potential glycosylation sites within the antigen binding domain were eliminated to prevent glycosylation that may interfere with antigen binding. See, e.g., U.S. Pat. No. 5,714,350.

b. Antibody Masking

In some embodiments, the antibodies disclosed herein are modified to limit their binding to specific cells and/or tissue. In one embodiment, such antibodies comprise a blocking peptide “mask” that specifically binds to the antigen binding surface of the antibody and interferes with antigen binding. In some embodiments, the mask is linked to each of the binding arms of the antibody by a protease cleavable linker. See, e.g., U.S. Pat. No. 8,518,404 to CytomX. Antibodies with protease cleavable linkers are useful for treatment of cancers in which protease levels are greatly increased in the tumor microenvironment compared with non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows disassociation of the masking/blocking peptide, enabling antigen binding selectively in the tumor, rather than in peripheral tissues in which antigen binding might cause unwanted side effects.

In another embodiment, a bivalent binding compound (“masking ligand”) comprising two antigen binding domains is developed that binds to both antigen binding surfaces of the (bivalent) antibody and interferes with antigen binding. In one embodiment, the two binding domain masks are linked to each other (but not the antibody) by a cleavable linker, for example, cleavable by a peptidase. (See, e.g., WO 2010/077643 to Tegopharm Corp). Masking ligands may comprise, or be derived from, the antigen to which the antibody is intended to bind, or may be independently generated (e.g., anti-idiotype binding fragments). Such masking ligands are useful for treatment of cancers in which protease levels are greatly increased in the tumor microenvironment compared with non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows disassociation of the two binding domains from each other, reducing the avidity for the antigen-binding surfaces of the antibody. The resulting dissociation of the masking ligand from the antibody enables antigen binding selectively in the tumor, rather than in peripheral tissues in which antigen binding might cause unwanted side effects.

c. Fcs and Modified Fc Regions

In one embodiment, the antibodies described herein may comprise Fc regions selected based on the biological activities of the antibody. Salfeld, Nat. Biotechnol. 25:1369 (2007). Human IgGs, for example, can be classified into four subclasses, IgG1, IgG2, IgG3, and IgG4. Each of these subclasses comprise an Fc region having a unique profile for binding to one or more of Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32a,c); FcγRIIIA and FcγRIIIB (CD16a,b) and inhibiting receptor FcγRIIB (CD32b), and for the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to FcγRIIA_(H131), and with lower affinity to FcγRIIA_(R131) FcγRIIIA_(V158); IgG4 binds to FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA_(V158); and the inhibitory receptor FcγRIIB has a lower affinity for IgG1, IgG2 and IgG3 than all other Fcγ receptors. (Bruhns et al. (2009) Blood 113:3716). Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Id. In general, with regard to ADCC activity, human IgG1≥IgG3>>IgG4≥IgG2. In some embodiments, an IgG1 constant domain, rather than an IgG2 or IgG4, is chosen, e.g., for use in a therapeutic composition because ADCC is desired.

Anti-hNKG2A antibody variable regions described herein may be linked (e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3 or IgG4 Fc, which may be of any allotype or isoallotype, e.g., for IgG1: G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v). (See, e.g., Jefferis et al. (2009) mAbs 1:1). Selection of allotype may be influenced by the potential immunogenicity concerns, e.g. to minimize the formation of anti-drug antibodies.

In some embodiments, anti-NKG2A antibodies of the present invention is unable to interact with human FcγRs. Since NKG2A is an inhibitory receptor expressed on CD8+ T and NK cells, avoiding or reducing agonism or depletion of NKG2A⁺ CD8+ T or NK cells enhances anti-tumor immunity. Thus, blockade of the NKG2A/HLA-E interaction was desired with an anti-NKG2A antibody unable to interact with human FcγRs.

d. Half-life Extension

In some embodiments, the anti-NKG2A antibody is modified to increase its biological half-life, e.g., the antibody's half-life in serum. Various approaches are known in the art. In one embodiment, the antibody is altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. For example, a combination Fc variant comprising M252Y, S254T and T256E, increases half-life-nearly four-fold. (Dall'Acqua et al. (2006) J Biol. Chem. 281:23514). Other modifications for increasing FcRn binding are described in Yeung et al. (2010) J. Immunol. 182:7663-7671; 6,277,375; 6,821,505; WO 97/34631; WO 2002/060919.

The serum half-life of the antibodies described herein can also be increased by pegylation. An antibody can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with a polyethylene glycol (PEG) reagent, such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. (See, e.g., EP 0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

In some instances, it may be desirable to decrease the half-life of an antibody, rather than to increase it. In some embodiments, the antibodies described herein include modifications to decrease their half-life. Modifications such as I253A (Hornick et al. (2000) J. Nucl. Med. 41:355) and H435A/R, I253A or H310A (Kim et al. (2000) Eur. J. Immunol. 29:2819) in Fc of human IgG1 can decrease FcRn binding, thus decreasing half-life (increasing clearance) for use in situations where rapid clearance is preferred, such as for medical imaging. (See also Kenanova et al. (2005) Cancer Res. 65:622). Other means to enhance clearance include formatting the antigen binding domains of the present invention as antibody fragments lacking the ability to bind FcRn, such as Fab fragments. Such modification can, for example, reduce the circulating half-life of an antibody from a couple of weeks to hours. Selective PEGylation of antibody fragments can then be used to increase the half-life of the antibody fragments when desired. (Chapman et al. (1999) Nat. Biotechnol. 17:780). Antibody fragments may also be fused to human serum albumin, e.g. in a fusion protein construct, to increase half-life. (Yeh et al. (1992) Proc. Nat'l Acad. Sci. 89:1904). Alternatively, a bispecific antibody may be constructed with a first antigen binding domain of the present invention and a second antigen binding domain that binds to human serum albumin (HSA). (See WO 2009/127691 and patent references cited therein). Alternatively, specialized polypeptide sequences can be added to antibody fragments to increase half-life, e.g. “XTEN” polypeptide sequences. (Schellenberger et al. (2009) Nat. Biotechnol. 27:1186; Int'l Pat. Appl. Pub. WO 2010/091122).

e. Additional Fc Variants

In some embodiments, when using an IgG1 constant domain, a potential protease cleavage site in the hinge of IgG1 constructs can be eliminated by D221G and K222S modifications, increasing the stability of the antibody. (WO 2014/043344).

The affinities and binding properties of an Fc variant for its ligands (Fc receptors) may be determined by a variety of in vitro assay methods (e.g., biochemical or immunological based assays) known in the art including but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE® SPR analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may use a label on one or more of the components being examined and/or employ various detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

In still other embodiments, the glycosylation of an antibody is modified to increase or decrease effector function. For example, an aglycoslated antibody can be made that lacks all effector function by mutating the conserved asparagine residue at position 297 (e.g. N297A), thus abolishing complement and FcγRI binding. (Bolt et al. (1993) Eur. J. Immunol. 23:403; see also Tao & Morrison (1989) J. Immunol. 143:2595 (using N297Q in IgG1 to eliminate glycosylation at position 297)).

Although aglycosylated antibodies generally lack effector function, mutations can be introduced to restore that function. Aglycosylated antibodies, e.g. those resulting from N297A/C/D/or H mutations or produced in systems (e.g. E. coli) that do not glycosylate proteins, can be further mutated to restore FcγR binding, e.g. S298G and/or T299A/G/or H (WO 2009/079242), or E382V and M4281 (Jung et al. (2010) Proc. Nat'l Acad. Sci. (USA) 107:604).

Glycoengineering can also be used to modify the anti-inflammatory properties of an IgG construct by changing the α2,6 sialyl content of the carbohydrate chains attached at Asn297 of the Fc regions, wherein an increased proportion of α2,6 sialylated forms results in enhanced anti-inflammatory effects. (See Nimmerjahn et al. (2008) Ann. Rev. Immunol. 26:513). Conversely, reduction in the proportion of antibodies having α2,6 sialylated carbohydrates may be useful in cases where anti-inflammatory properties are not wanted. Methods of modifying α2,6 sialylation content of antibodies, for example, by selective purification of α2,6 sialylated forms or by enzymatic modification, are provided at U.S. Pat. Appl. Pub. No. 2008/0206246. In other embodiments, the amino acid sequence of the Fc region may be modified to mimic the effect of α2,6 sialylation, for example, by inclusion of an F241A modification. (WO 2013/095966).

III. Antibody Physical Properties

In certain embodiments, the antibodies described herein contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or an altered antibody pharmacokinetics due to altered antigen binding (Marshall et al (1972) Ann. Rev. Biochem. 41:673-702; Gala and Morrison (2004) J. Immunol. 172:5489-94; Wallick et al (1988) J. Exp. Med. 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation has been known to occur at motifs containing an N-X-S/T sequence. In some embodiments, the anti-hNKG2A antibody does not contain variable region glycosylation. Such antibodies can be obtained by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues within the glycosylation region.

In certain embodiments, the antibodies described herein do not contain asparagine isomerism sites. The deamidation of asparagine may occur on N-G or D-G sequences and result in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and decreases its stability (known as the isoaspartic acid effect).

In some embodiments, the antibodies described herein have an isoelectric point (pI) in the pH range between 6 and 9.5. In some embodiments, the antibodies described herein have a pI in the pH range of 7-9.5 or 6-8. Antibodies having a pI within a desired pI range can be obtained either by selecting antibodies with a pI in the pH range from a group of candidates or by mutating charged surface residues of a particular antibody.

In some embodiments, the antibodies described herein are selected and/or engineered have a temperature of initial unfolding (T_(M1)) greater than 60° C., greater than 65° C., or greater than 70° C. The melting point of an antibody may be measured using differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett. 68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr. Sci. 40:343-9).

In some embodiments, the antibodies described herein are selected and/or engineered to have advantageous degradation properties, e.g., slow degradation in vitro and/or in vivo. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995) Anal Chem. 67:3626-32). In some embodiments, the antibodies described herein are selected and/or engineered to have favorable aggregation properties, e.g., antibodies that show minimal aggregation in vitro and/or in vivo, which may elicit an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. In some embodiments, the antibodies described herein show aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less compared to aggregation of the parent antibody. Aggregation can be measured by several techniques, including size-exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.

IV. Nucleic Acid Molecules and Recombinant Methods

Another aspect described herein pertains to nucleic acid molecules that encode the anti-hNKG2A antibodies described herein. The nucleic acids may be present in whole cells e.g., a host cell, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., the chromosomal DNA that is linked to the isolated DNA in nature) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well known in the art. (See, F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York). A nucleic acid described herein can be, for example, DNA or RNA and may or may not contain introns. In a certain embodiments, the nucleic acid is a cDNA molecule.

Nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and/or heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term “operatively linked,” as used in this context, means that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

Isolated DNA encoding the V_(H) region can be converted to a full-length heavy chain gene by operatively linking the V_(H)-encoding DNA to another DNA molecule encoding heavy chain constant regions (hinge, CH1, CH2 and/or CH3). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, et al., 1991), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG (IgG1, IgG2, IgG3, or IgG4), IgA, IgE, IgM or IgD constant region, for example, an IgG1 region. For a Fab fragment heavy chain gene, the V_(H)-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the V_(L)-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat, et al., 1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃ (SEQ ID NO: 160), such that the V_(H) and V_(L) sequences can be expressed as a contiguous single-chain protein, with the V_(L) and V_(H) regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

V. Antibody Generation

Various antibodies of the present invention, e.g. those that bind to the same epitope as selected anti-hNKG2A antibodies disclosed herein, can be produced using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies also can be employed, e.g., viral or oncogenic transformation of B lymphocytes, phage display technique using libraries of human antibody genes.

An exemplary animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies described herein can be prepared based on the sequence of a murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody, the murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

In one embodiment, the antibodies described herein are human monoclonal antibodies. Such human monoclonal antibodies directed against human NKG2A can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode un-rearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGκ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparation and use of HuMab mice, and the genomic modifications carried by such mice, is further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of all of which are hereby specifically incorporated by reference in their entirety. (See, also, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 to Korman et al.)

In certain embodiments, antibodies described herein are raised using a mouse that carries human immunoglobulin sequences on transgenes and transchromosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as “KM mice”, are described in detail in PCT Publication WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-hNKG2A antibodies described herein. For example, an alternative transgenic system referred to as the Xenomouse (Abgenix, Inc.) can be used; such mice are described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-nkg2a antibodies described herein. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as “TC mice” can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. Furthermore, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to raise anti-hNKG2A antibodies described herein.

Additional mouse systems described in the art for raising human antibodies, e.g., human anti-hNKG2Aantibodies, include (i) the VELOCIMMUNE® mouse (Regeneron Pharmaceuticals, Inc.), in which the endogenous mouse heavy and light chain variable regions have been replaced, via homologous recombination, with human heavy and light chain variable regions, operatively linked to the endogenous mouse constant regions, such that chimeric antibodies (human V/mouse C) are raised in the mice, and then subsequently converted to fully human antibodies using standard recombinant DNA techniques; and (ii) the MeMo® mouse (Merus Biopharmaceuticals, Inc.), in which the mouse contains un-rearranged human heavy chain variable regions but a single rearranged human common light chain variable region. Such mice, and use thereof to raise antibodies, are described in, for example, WO 2009/15777, US 2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO 2011/163314, WO 2012/148873, US 2012/0070861 and US 2012/0073004.

Human monoclonal antibodies described herein can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art. (See, e.g., U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat. No. 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.).

Human monoclonal antibodies described herein can also be prepared using mice with severe combined immunodeficiency (SCID) into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

Immunizations

To generate fully human antibodies to human NKG2A, mice or transgenic or transchromosomal mice containing human immunoglobulin genes (e.g., HCo12, HCo7 or KM mice) can be immunized with a purified or enriched preparation of the NKG2A antigen and/or cells expressing NKG2A, as described for other antigens, for example, by Lonberg et al. (1994) Nature 368(6474): 856-859; Fishwild et al. (1996) Nature Biotechnology 14: 845-851 and WO 98/24884. Alternatively, mice can be immunized with DNA encoding human NKG2A. Preferably, the mice will be 6-16 weeks of age upon the first infusion. For example, a purified or enriched preparation (e.g, 5 μg-50 μg) of the recombinant human NKG2A antigen can be used to immunize the mice intraperitoneally. If the immunizations using a purified or enriched preparation of the NKG2A antigen do not result in antibodies, mice can also be immunized with cells expressing NKG2A, e.g., a cell line, to promote immune responses.

The HuMAb transgenic mice can be initially immunized intraperitoneally or subcutaneously (SC) with antigen in Ribi's adjuvant, followed by every other week IP/SC immunizations (up to a total of 10) with antigen in Ribi's adjuvant. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened by ELISA and FACS (as described below), and mice with sufficient titers of anti-NKG2A human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen three days before sacrifice and removal of the spleen and lymph nodes. Two to three fusions for each immunization may be performed. Between 6 and 24 mice can be immunized for each antigen. In some embodiments, HCo7, HCo12, and KM strains are used. In addition, both HCo7 and HCo12 transgene can be bred together into a single mouse having two different human heavy chain transgenes (HCo7/HCo12).

Generation of Hybridomas Producing Monoclonal Antibodies to NKG2A Protein

To generate hybridomas producing monoclonal antibodies described herein, splenocytes and/or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to Sp2/0 non-secreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG. Cells are plated at approximately 2×10⁵ in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 10% fetal Clone Serum, 18% “653” conditioned media, 5% Origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma). After approximately two weeks, cells can be cultured in medium in which the HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium can be observed usually after 10-14 days. The antibody secreting hybridomas can be re-plated, screened again, and, if still positive for human IgG, the monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

To purify monoclonal antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at −80° C.

VI. Antibody Manufacture

Generation of Transfectomas Producing Monoclonal Antibodies to NKG2A

Antibodies of the present invention, including both specific antibodies for which sequences are provided and other, related anti-NKG2A antibodies, can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (Morrison, S. (1985) Science 229:1202).

For example, to express antibodies, or antibody fragments thereof, DNAs encoding partial or full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest), and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector(s) by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the V_(H) segment is operatively linked to the C_(H) segment(s) within the vector and the V_(L) segment is operatively linked to the C_(L) segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, recombinant expression vectors may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term “regulatory sequence” includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, amongst other factors. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP), and polyomavirus. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or β-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr− host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term “transfection” encompasses a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies described herein in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for production of high yields of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6:12-13). Antibodies of the present invention can also be produced in glycol-engineered strains of yeast. (Pichia pastoris. Li et al. (2006) Nat. Biotechnol. 24:210).

Exemplary mammalian host cells for expressing the recombinant antibodies described herein include CHO cells (including dhfr− CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a dihydrofolate reductase (DHFR) selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982)Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another exemplary expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

The N- and C-termini of antibody polypeptide chains of the present invention may differ from the expected sequence due to commonly observed post-translational modifications. For example, C-terminal lysine residues are often missing from antibody heavy chains. (Dick et al. (2008) Biotechnol. Bioeng. 100:1132). N-terminal glutamine residues, and to a lesser extent glutamate residues, are frequently converted to pyroglutamate residues on both light and heavy chains of therapeutic antibodies. (Dick et al. (2007) Biotechnol. Bioeng. 97:544; Liu et al. (2011) JBC 28611211; Liu et al. (2011) J. Biol. Chem. 286:11211).

Amino acid sequences for various anti-hNKG2A antibodies of the present invention are provided in the Sequence Listing. For the reasons discussed above, the C-terminal lysine is not included in many of sequences in the Sequence Listing for heavy chains or heavy chain constant domains. However, in an alternative embodiment, each heavy chain for the anti-hNKG2A antibodies of the present invention, and/or genetic construct encoding such antibodies or the heavy or light chains thereof, includes this additional lysine residue at the C-terminus of the heavy chain(s).

VII. Assays

Antibodies described herein can be tested for binding to NKG2A by, for example, standard ELISA. For example, microtiter plates are coated with purified NKG2A at 1-2 μg/ml in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from NKG2A-immunized mice) are added to each well and incubated for 1-2 hours at 37° C. The plates are washed with PBS/Tween and then incubated with secondary reagent (e.g., for human antibodies, or antibodies otherwise having a human heavy chain constant region, a goat-anti-human IgG Fc-specific polyclonal reagent) conjugated to horseradish peroxidase (HRP) for 1 hour at 37° C. After washing, the plates are developed with ABTS substrate (Moss Inc, product: ABTS-1000) and analyzed by a spectrophotometer at OD 415-495. Sera from immunized mice are then further screened by flow cytometry for binding to a cell line expressing human NKG2A, but not to a control cell line that does not express NKG2A. Briefly, the binding of anti-NKG2A antibodies is assessed by incubating NKG2A expressing CHO cells with the anti-NKG2A antibody at 1:20 dilution. The cells are washed and binding is detected with a PE-labeled anti-human IgG Ab. Flow cytometric analyses are performed using a FACScan flow cytometry (Becton Dickinson, San Jose, Calif.). Preferably, mice that develop the highest titers will be used for fusions. Analogous experiments may be performed using anti-mouse detection antibodies if mouse anti-HNKG2A antibodies are to be detected.

An ELISA, e.g., as described above can be used to screen for antibodies and, thus, hybridomas that produce antibodies that show positive reactivity with the NKG2A immunogen. Hybridomas that produce antibodies that bind, preferably with high affinity, to NKG2A can then be subcloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA), can then be chosen for making a cell bank, and for antibody purification.

To purify anti-NKG2A antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at −80° C.

To determine if the selected anti-NKG2A monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.). Biotinylated MAb binding can be detected with a streptavidin labeled probe. Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using NKG2A coated-ELISA plates as described above.

To determine the isotype of purified antibodies, isotype ELISAs can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, wells of microtiter plates can be coated with 1 μg/ml of anti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA, the plates are reacted with 1 μg/ml or less of test monoclonal antibodies or purified isotype controls, at ambient temperature for one to two hours. The wells can then be reacted with either human IgG1 or human IgM-specific alkaline phosphatase-conjugated probes. Plates are developed and analyzed as described above.

To test the binding of monoclonal antibodies to live cells expressing NKG2A, flow cytometry can be used. Briefly, cell lines expressing membrane-bound NKG2A (grown under standard growth conditions) are mixed with A concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4° C. for one hour. After washing, the cells are reacted with Phycoerythrin (PE)-labeled anti-IgG antibody under the same conditions as the primary antibody staining. The samples can be analyzed by FACScan instrument using light and side scatter properties to gate on single cells and binding of the labeled antibodies is determined. An alternative assay using fluorescence microscopy may be used (in addition to or instead of) the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but may have diminished sensitivity depending on the density of the antigen.

Anti-hNKG2A antibodies can be further tested for reactivity with the NKG2A antigen by Western blotting. Briefly, cell extracts from cells expressing NKG2A can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked with 20% mouse serum, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

Methods for analyzing binding affinity, cross-reactivity, and binding kinetics of various anti-NKG2A antibodies include standard assays known in the art, for example, Biolayer Interferometry (BLI) analysis, and Biacore SPR analysis using a Biacore SPR instrument.

In one embodiment, an anti-hNKG2A antibody specifically binds to the extracellular region of human NKG2A. In one embodiment, the antibody binds to a particular domain (e.g., a functional domain) within the extracellular domain of NKG2A. In one embodiment, the anti-hNKG2A antibody specifically binds to the extracellular region of human NKG2A and the extracellular region of cynomolgus NKG2A. In one embodiment, the anti-hNKG2A antibody binds to human NKG2A with high affinity.

VIII. Multispecific Molecules

In certain embodiments, antibodies described herein may be multispecific, e.g., bispecific or trispecific, molecules. Multispecific antigen-binding molecules, such as multispecific antibodies, comprise two or more antigen-binding site, each specific for a different epitope. The different epitope can be part of the same or different antigens. In one embodiment, one antigen-binding site is specific for human NKG2A and the other for a different antigen. In one embodiment, an anti-h NKG2A antibody, or antigen-binding fragments thereof, as described herein is linked to another antigen-binding molecule, e.g., another peptide or protein (e.g., another antibody or antibody fragment, or a ligand for a receptor) having a different binding specificity to generate a bispecific molecule that binds to at least two different binding sites or target molecules. In one embodiment, the antibody described herein is derivatized or linked to more than one other antigen-binding molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules. Accordingly, provided herein are bispecific molecules comprising at least one first binding specificity for NKG2A and a second binding specificity for a second target epitope. In an embodiment described herein in which the bispecific molecule is multispecific, the molecule can further include a third binding specificity.

In one embodiment, the bispecific molecules described herein comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Pat. No. 4,946,778, the contents of which is expressly incorporated by reference.

While human monoclonal antibodies are preferred, other antibodies that can be employed in the bispecific molecules described herein are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules described herein can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule has a combination of binding specificities such as a (mAb×mAb), (mAb×Fab), (Fab×F(ab′)₂) or (ligand x Fab) fusion protein. A bispecific molecule described herein can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may comprise at least two single chain molecules. Methods for preparing bispecific molecules are described for example in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can be confirmed using art-recognized methods, such as using ELISA, radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

IX. Compositions

Further provided are compositions, e.g., a pharmaceutical compositions, containing one or more anti-NKG2A antibodies, or antigen-binding fragment(s) thereof, as described herein, formulated together with a pharmaceutically acceptable carrier. Accordingly, the compositions of the present invention include the human or humanized anti-hNKG2A antibodies (or antigen-binding fragments thereof) having the CDR sequences, the heavy and/or light chain variable region sequences, or the full-length heavy and/or light chain sequences set forth herein. Compositions of the present invention also include anti-hNKG2A antibodies having sequences which are variants of the sequences set forth in the Sequence Listing. For example, such antibodies can comprise sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, or 99% identical to the CDR sequences, the heavy and/or light chain variable region sequences, or full-length heavy and/or light chain sequences set forth in Sequence Listing.

Such compositions also may include one or a combination of (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules described herein. For example, a pharmaceutical composition described herein can comprise a combination of antibodies (or immunoconjugates or bispecific antibodies) that bind to different epitopes on the target antigen or that have complementary activities.

Pharmaceutical compositions described herein also can be administered as combination therapy, i.e., anti-NKG2A antibodies combined with other agents. For example, the combination therapy can include an anti-NKG2A antibody described herein combined with at least one other anti-cancer and/or T-cell stimulating (e.g., activating) agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the antibodies described herein.

In some embodiments, pharmaceutical compositions disclosed herein can include other compounds, drugs, and/or agents used for the treatment of cancer. Such compounds, drugs, and/or agents can include, for example, chemotherapy drugs, small molecule drugs or antibodies that stimulate the immune response to a given cancer. In some embodiments, a pharmaceutical composition comprises a first antibody specific for anti-h NKG2A and a second antibody.

In some embodiments, the first antibody and the second antibody are present in the composition at a fixed dose (i.e., a fixed ratio). In other embodiments, this fixed dose is between at least about 1:200 to at least about 200:1, at least about 1:150 to at least about 150:1, at least about 1:100 to at least about 100:1, at least about 1:75 to at least about 75:1, at least about 1:50 to at least about 50:1, at least about 1:25 to at least about 25:1, at least about 1:10 to at least about 10:1, at least about 1:5 to at least about 5:1, at least about 1:4 to at least about 4:1, at least about 1:3 to at least about 3:1, or at least about 1:2 to at least about 2:1 mg anti-hNKG2A antibody to mg second antibody. In some embodiments, the fixed dose is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, or about 1:200 anti-h NKG2A antibody to second antibody. In some embodiments, the fixed dose is at least about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 120:1, about 140:1, about 160:1, about 180:1, or about 200:1 mg first antibody to mg second antibody. For example, in one embodiment, the anti-h NKG2A antibody and the second antibody are administered as described in the Examples.

The additional antibodies include, for example, one or more of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-OX40 (also known as CD134, TNFRSF4, ACT35 and/or TXGP1L) antibody, an anti-LAG-3 antibody, an anti-CD73 antibody, an anti-CD137 antibody, an anti-CD27 antibody, or an anti-CSF-1R antibody.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In some embodiments, the carrier is suitable for intravenous administration. In other embodiments, the carrier is suitable for subcutaneous administration. In some embodiments, the composition comprising anti-NKG2A antibody is delivered subcutaneously using Halozyme's ENHANZE® drug delivery technology, which includes a recombinant human hyaluronidase enzyme (rHuPH20) that temporarily degrades hyaluronan. In some embodiments, the ENHANZE® drug delivery technology allows for subcutaneous administrations of compositions that is more rapid as compared to intravenous administration. In other embodiments, depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977)J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition described herein also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX™, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions described herein is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those described above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Out of one hundred percent, this amount may range from about 0.01 percent to about ninety-nine percent of active ingredient, e.g., from about 0.1 percent to about 70 percent, e.g., from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

In some embodiments, the composition includes an anti-NKG2A antibody, such as the NKG2A.9. The composition is a sterile, non-pyrogenic, single-use, preservative-free, isotonic aqueous solution for intravenous administration. The composition may be administered undiluted or further diluted with 0.9% sodium chloride injection to the required protein concentrations prior to infusion. In some embodiments, the anti-NKG2A antibody includes the following excipients: L-histine, L-histidine hydrochloride monohydrate, sucrose, pentetic acid (also known as diethylenetriaminepentaaceitc acid, polysorbate 80, and water for the injection.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms described herein are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

For administration of the antibody, the dosage may range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example, dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. Alternatively, administration of the antibody is a flat dose which may range from 2 mg to 800 mg, for example, a dose of 25 mg, 80 mg, 200 mg, or 400 mg. An exemplary treatment regimen entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, once every five months, or once every six months. In some embodiments, the treatment regimen includes an initial dose, and then a maintenance dose of a different dose amount at an intermittent dose interval.

In some embodiments, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. In some embodiments, the therapeutic antibody is administered on multiple occasions. Intervals between single dosages can be, for example, weekly, once every three weeks, once every four weeks, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some embodiments, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg/ml and in some methods about 25-300 μg/ml.

In some embodiments, the antibody can be administered as a sustained release formulation. Administration via a sustained release formulations might require less frequent administration. Dosage and frequency vary depending on the half-life of the antibody in the patient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In some embodiments, a relatively high dosage at relatively short intervals is administered for therapeutic treatment. In some embodiments, a relatively high dosage is administered until progression of the disease is reduced or terminated, e.g., until the patient shows partial or complete amelioration of symptoms of disease. In some embodiments, a prophylactic treatment is administered to patient following a therapeutic treatment.

Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-NKG2A antibody described herein preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. In the context of cancer, a therapeutically effective dose preferably prevents further deterioration of physical symptoms associated with cancer. Symptoms of cancer are well-known in the art and include, for example, unusual mole features, a change in the appearance of a mole, including asymmetry, border, color and/or diameter, a newly pigmented skin area, an abnormal mole, darkened area under nail, breast lumps, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, worsening breathlessness, coughing up blood, blood in the urine, blood in stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal fullness, bloating, fluid in peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of colon, acute peritonitis (infection, fever, pain), pain, vomiting blood, heavy sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle spasms, colon metastases, lung metastases, bladder metastases, liver metastases, bone metastases, kidney metastases, and pancreatic metastases, difficulty swallowing, and the like. Therapeutic efficacy may be observable immediately after the first administration of an anti-hNKG2A monoclonal antibody of the present invention, or it may only be observed after a period of time and/or a series of doses. Such delayed efficacy my only be observed after several months of treatment, e.g., up to 6, 9 or 12 months.

A therapeutically effective dose may prevent or delay onset of cancer, such as may be desired when early or preliminary signs of the disease are present. Accordingly, any clinical or biochemical assay that monitors any of the foregoing may be used to determine whether a particular treatment is a therapeutically effective dose for treating cancer. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

A composition described herein can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Exemplary routes of administration for antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.

Alternatively, an antibody described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition described herein can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use with anti-hNKG2A antibodies described herein include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the anti-hNKG2A antibodies described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995)Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

Also within the scope described herein are kits comprising the antibody compositions described herein (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. The kit can further contain at least one additional reagent, or one or more additional human antibodies described herein. Kits can include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or that otherwise accompanies the kit.

X. Methods of Use

The antibodies, antibody compositions and methods described herein have numerous in vitro and in vivo uses involving, for example, enhancement of immune response by blocking the NKG2A/HLA-E interaction. In one embodiment, the anti-NKG2A antibodies described herein are monoclonal human or humanized antibodies. In one embodiment, anti-hNKG2A antibodies described herein (e.g., 13F3.A4, NKG2A.9, and NKG2A.11) can be administered to cells in culture, in vitro or ex vivo, or to human subjects to enhance immunity in a variety of diseases. In a particular embodiment, the anti-hNKG2A antibodies are antagonistic antibodies. Provided herein are methods of modifying an immune response in a subject comprising administering to the subject an anti-NKG2A antibody, or antigen-binding fragment thereof, described herein such that the immune response in the subject is enhanced, stimulated or up-regulated. In one embodiment, administering the anti-hNKG2A antibody according to the methods described herein enhances T cell and/or NK cell responses. In one embodiment, administering the anti-hNKG2A antibody according to the methods described herein stimulates, enhances or upregulates antigen-specific T cell responses to a tumor. The T cells can be Teff cells, e.g., CD4+ Teff cells, CD8+ Teff cells, T helper (T_(h)) cells and T cytotoxic (T_(a)) cells. A tumor may be a solid tumor or a liquid tumor, e.g., a hematological malignancy. In certain embodiments, a tumor is an immunogenic tumor. In certain embodiments, a tumor is non-immunogenic. In certain embodiments, a tumor is PD-L1 positive. In certain embodiments a tumor is PD-L1 negative. A subject may also be a virus-bearing subject and an immune response against the virus is enhanced. In one embodiment, administering the anti-hNKG2A antibody according to the methods described herein stimulates, enhances or upregulates NK cell responses.

In one embodiment, the methods result in an enhancement of an immune response in a human subject wherein such enhancement has a desirable effect. In one embodiment, the human subject is a human patients having a disorder that can be treated by augmenting an immune response, e.g., the T-cell mediated immune response. In a particular embodiment, the human patient has a cancer. In one embodiment, the anti-hNKG2A antibodies described herein can be administered together with an antigen of interest or the antigen may already be present in the subject to be treated, e.g., a tumor-bearing or virus-bearing subject. When anti-NKG2A antibodies are administered together with another agent, the two can be administered separately or simultaneously.

Further provided are methods for inhibiting growth of a tumor cell in a subject comprising administering to the subject an anti-hNKG2A antibody described herein such that growth of the tumor cell is inhibited in the subject, such as a human subject. Also provided are methods of treating chronic viral infection in a subject comprising administering to the subject an anti-NKG2A antibody described herein such that the chronic viral infection is treated in the subject, such as a human subject.

In some embodiments, an anti-NKG2A antibody is administered to a subject, e.g., a human patient, as an adjunctive therapy, adjuvant therapy, or neo-adjuvant therapy. In some embodiments, treatments of subjects having cancer with an anti-NKG2A antibody may lead to a long-term durable response relative to the current standard of care; long term survival of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years, recurrence free survival of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more years. In certain embodiments, treatment of a subject having cancer with an anti-hNKG2A antibody prevents recurrence of cancer or delays recurrence of cancer by, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more years. An anti-NKG2A treatment can be used as a first, second, or subsequent line of treatment.

These and other methods described herein are discussed in further detail below.

Cancer

Provided herein are methods for treating a subject having cancer, comprising administering to the subject an anti-hNKG2A antibody described herein, such that the subject is treated, e.g., such that growth of cancerous tumors is inhibited or reduced and/or that the tumors regress. An anti-NKG2A antibody can be used alone to inhibit the growth of cancerous tumors. Alternatively, an anti-NKG2A antibody can be used in conjunction with another agent, e.g., other immunogenic agents, standard cancer treatments, or other antibodies, as described below. Combination with an inhibitor of PD-1, such as an anti-PD-1 or an anti-PD-L1 antibody, is also provided. Combination with an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody, is also provided. Combination with an inhibitor of PD-1 and an inhibitor of CTLA-4 is also provided. Combination with an agonist antibody of ICOS is also provided.

In one aspect, provided herein are methods of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an anti-NKG2A antibody described herein. In one embodiment, the anti-NKG2A antibody may be a chimeric antibody, a human antibody, or a humanized anti-NKG2A antibody. In one embodiment, the methods of treating a cancer described herein comprise administering an anti-NKG2A antibody that contacts human NKG2A at one or more amino acid residues of:

(a) (SEQ ID NO: 156) LSIDNEEMKF; (b) (SEQ ID NO: 157) PSSWIGVFRNSSHHPW; (c) (SEQ ID NO: 158) LAFKHEIKDSDN; and (d) (SEQ ID NO: 159) QVNRLKSAQQCGSSIIYHC.

In another embodiment, the methods of treating a cancer described herein comprise administering an anti-NKG2A antibody that contacts human NKG2A at one or more amino acid residues of:

(a) (SEQ ID NO: 156) LSIDNEEMKF; (b) (SEQ ID NO: 157) PSSWIGVFRNSSHHPW; (c) (SEQ ID NO: 158) LAFKHEIKDSDN; (d) L; and (e) (SEQ ID NO: 159) QVNRLKSAQQCGSSIIYHC.

In another embodiment, the method comprises administering the NKG2A.9 antibody to treat cancer. In another embodiment, the method comprises administering a composition comprising the 13F3.A4 antibody to treat cancer. In another embodiment, the method comprises administering the NKG2A.11 antibody to treat cancer. In another embodiment, the method comprises administering a composition comprising the NKG2A.9 antibody to treat cancer. In another embodiment, the method comprises administering a composition comprising the NKG2A.11 antibody to treat cancer. In another embodiment, the method comprises administering the 13F3.A4 antibody or a variant thereof to treat cancer. In another embodiment, the method comprises administering a composition comprising the 13F3.A4 antibody or a variant thereof to treat cancer.

Examples of cancer include, but are not limited to, squamous cell carcinoma, small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain cancer including brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally-induced cancers including those induced by asbestos, virus-related cancers (e.g., human papilloma virus (HPV)-related tumor), and hematologic malignancies derived from either of the two major blood cell lineages, i.e., the myeloid cell line (which produces granulocytes, erythrocytes, thrombocytes, macrophages, and mast cells) or lymphoid cell line (which produces B, T, NK, and plasma cells), such as all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic and/or myelogenous leukemias, such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplantation lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmocytoma, and multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nervous, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma, hematopoietic tumors of lymphoid lineage, for example T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) preferably of the T-cell type; a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head or neck, renal cancer, rectal cancer, cancer of the thyroid gland; acute myeloid lymphoma, as well as any combinations of said cancers. In one embodiment, the methods described herein may also be used for treatment of metastatic cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, e.g., with a blocking CTLA-4 and/or PD-1 antibody), and recurrent cancers.

In one embodiment, the anti-hNKG2A antibody is administered as a monotherapy. In one embodiment, the anti-hNKG2A agonist antibody is administered as the only immunostimulating agent. In one embodiment, the anti-hNKG2A is administered to a patient with another agent. In one embodiment, an anti-hNKG2A antibody is administered with an immunogenic agent. In one embodiment, the anti-hNKG2A antibody is administered in conjunction with a cancer vaccine. In some embodiments, the cancer vaccine comprises cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines (He et al. (2004) J. Immunol. 173:4919-28). In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments the peptide cancer vaccine is a multivalent long peptide, a multi-peptide, a peptide cocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some embodiments, an anti-hNKG2A antibody is administered in conjunction with an adjuvant. Non-limiting examples of tumor vaccines that are used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination. Dranoff et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 3539-43.

Other cancer vaccines are the proteins from viruses implicated in human cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). In some embodiments, another form of tumor specific antigen that is used with NKG2A inhibition is purified heat shock proteins (HSP) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins from the tumor cells and these HSPs are highly efficient at delivery to antigen presenting cells for eliciting tumor immunity (Suot & Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

In some embodiments, dendritic cells are potent antigen presenting cells that are used to prime antigen-specific responses. Dendritic cells can be produced ex vivo and loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). Dendritic cells can also be transduced by genetic means to express these tumor antigens as well. DCs have also been fused directly to tumor cells for the purposes of immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As a method of vaccination, Dendritic cell immunization can be effectively combined with anti-NKG2A antibodies to activate (unleash) more potent anti-tumor responses.

In some embodiments, an anti-hNKG2A is administered with standard of care treatment, e.g., surgery, radiation, and/or chemotherapy. In some embodiments, an anti-hNKG2A antibody is administered with a chemotherapeutic agent. In some embodiments, the anti-hNKG2A antibody is administered with one or more of carboplatin, cisplatin, paclitaxel, nab-paclitaxel, gemcitabine or FOLFOX. In some embodiment, an anti-hNKG2A antibody is administered with carboplatin or nab-paclitaxel. In some embodiments, an anti-hNKG2A antibody is administered in conjunction with carboplatin and paclitaxel. In some embodiments, an anti-hNKG2A antibody is administered with cisplatin and pemetrexed. In some embodiments, an anti-hNKG2A antibody is administered with cisplatin and gemcitabine. In some embodiments, an anti-hNKG2A antibody is administered with FOLFOX. In some embodiments, an anti-hNKG2A antibody is administered with FOLFIRI. In one embodiment, an anti-hNKG2A antibody is administered with dacarbazine for the treatment of melanoma. In some embodiments, cisplatin is intravenously administered as a 100 mg/ml dose once every four weeks. In some embodiments, an anti-hNKG2A antibody is administered in with doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine and/or cyclophosphamide hydroxyurea. In some embodiments, adriamycin is intravenously administered as a 60 mg/ml to 75 mg/ml dose once every 21 days. In one embodiment, the anti-hNKG2A antibody is administered to a human patient that is resistant to treatment with at least one drug, wherein administration of the anti-hNKG2A antibody reduces, alleviates, or abrogates resistance to the at least one drug. In some embodiments, an anti-hNKG2A antibody is administered with an agonist antibody, such as an anti-ICOS antibody.

The combination therapies described above can be administered in various combinations with each other, and encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. Antibodies of the invention can also be used in combination with radiation therapy.

In some embodiments, another example of such a combination is an anti-hNKG2A antibody administered antibody in combination with interleukin-2 (IL-2). In some embodiments, the combination of anti-hNKG2A antibody and IL-2 is to treat various cancers, including for the treatment of renal cell carcinoma and melanoma. In some embodiments, the anti-hNKG2A antibodies discussed herein is combined with an IL-2 pathway agonist to treat various cancers. The combination includes various IL-2 pathway agonists, such as those described in WO 2012/065086 (Nektar Therapeutics) and WO 2015/125159 (Nektar Therapeutics), the contents of which are incorporated by reference in their entireties. WO 2006/138572 (Nektar Therapeutics) provides conjugates having a degradable linkage and polymeric reagents useful in preparing such conjugates, as well as methods of making polymeric reagents and conjugates, and is incorporated by reference in its entirety.

In some embodiments, the combination of an anti-hNKG2A antibody as described herein, such as NKG2A.9, NKG2A.11, or 13F3.A4 antibodies, and an IL-2 pathway agonist, such as NKTR-214, is administered to patients to treat cancer. As described in more detail below, NKTR-214 is produced by conjugating on average around six FMOC (fluorenylmethyloxycarbonyl chloride)-based polyethylene glycol (PEG) reagents having the following structure (mPEG₂-C2-fomc-20K-N-Hydroxysuccinimidate Derivative, 20 kDa, (“mPEG2-C2-fmoc-20K-NHS”):

to a protein having the following 132-amino acid sequence:

(SEQ ID NO: 161) PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKELQCLEE  60 ELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW 120 ITFSQSIISTLT 132 

WO 2012/065086 provides conjugates of an IL-2 moiety and one or more non-peptide, water-soluble polymers, including polyethylene glycol or a derivative thereof. Specifically, Example 2 (paragraphs 202-204) of WO 2012/065086 describes PEGylation of rIL-2 with mPEG2-C2-fmoc-20K-NHS to result in the mPEG2-C2-fmoc-20K-NHS structure set forth above. Example 1 (paragraphs 63-66) WO 2015/125159 describes a scaled-up approach for PEGylating IL-2 with mPEG2-C2-fmoc-20K-NHS that results in RSLAIL-2 (NKTR-214). NKTR-214 is a cytokine that is designed to target CD122, (also known as interleukin-2 receptor beta subunit, IL-2β), a protein found on certain immune cells (e.g., CD8+ T Cells and NK Cells), to expand these cells to promote their anti-tumor effects.

In some embodiments, an anti-hNKG2A antibody is administered in combination with an anti-angiogenic agent.

Other combination therapies that may result in synergy with the anti-hNKG2A antibodies described herein through cell death are radiation, surgery, and hormone deprivation.

In some embodiments, anti-hNKG2A antibodies described herein is administered in conjunction with bispecific antibodies. Bispecific antibodies can be used to target two different antigens. In some embodiments, anti-hNKG2A antibodies are used in combination with bispecific antibodies that target Fcα or Fcγ receptor-expressing effectors cells to treat tumor cells (see, e.g. U.S. Pat. Nos. 5,922,845 and 5,837,243). For example, anti-Fc receptor/anti-tumor antigen (e.g., Her-2/neu) bispecific antibodies have been used to target macrophages to sites of tumor. In some embodiments, the T cell arm of these responses is augmented by the functional activity of the anti-hNKG2A antibody. In some embodiments, antigen is delivered directly to DCs by the use of bispecific antibodies that bind to tumor antigen and a dendritic cell specific cell surface marker. In some embodiments, anti-hNKG2A antibodies are used in combination with antibodies that reduce or inactivate the immunosuppressive proteins expressed by a tumor, e.g., anti-TGF-β antibodies, anti-IL-10 antibodies, and anti-Fas ligand antibodies.

Infectious Diseases

In another aspect, the invention described herein provides a method of treating an infectious disease in a subject, including a human subject, comprising administering to the subject an anti-hNKG2A antibody, or antigen-binding fragment thereof, such that the subject is treated for the infectious disease. In other embodiments, the anti-NKG2A antibody is a chimeric or humanized antibody.

Similar to its treatment of tumors as discussed herein, anti-hNKG2A antibodies described herein can be administered alone, or as an adjuvant, in combination with vaccines, to enhance the immune response to pathogens, toxins, and self-antigens, including to treat chronic viral infections. Examples of pathogens for which this therapeutic approach can be particularly useful, include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are less than completely effective. These pathogens include, but are not limited to HIV, hepatitis (A, B, & C), influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.

Examples of pathogenic viruses causing infections treatable by methods described herein include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

Examples of pathogenic bacteria causing infections treatable by methods described herein include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria.

Examples of pathogenic fungi causing infections treatable by methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Examples of pathogenic parasites causing infections treatable by methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis.

The methods described herein of administering anti-hNKG2A antibodies to a subject may be combined with other forms of immunotherapy such as cytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which provides for enhanced presentation of tumor antigens (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).

Autoimmune Reactions

In some aspects, Anti-NKG2A antibodies increase autoimmune responses. Induction of anti-tumor responses using tumor cell and peptide vaccines reveals that many antitumor responses involve anti-self reactivities (van Elsas et al. (2001) J. Exp. Med. 194:481-489; Overwijk, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987; Hurwitz, (2000) supra; Rosenberg & White (1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4). Therefore, anti-NKG2A antibodies are used with various self proteins to devise vaccination protocols to efficiently generate immune responses against these self proteins for disease treatment. For example, Alzheimer's disease involves inappropriate accumulation of Aβ peptide in amyloid deposits in the brain; antibody responses against amyloid are able to clear these amyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other self proteins can also be used as targets such as IgE for the treatment of allergy and asthma, and TNF-α for rheumatoid arthritis. Finally, antibody responses to various hormones can be induced by the use of anti-NKG2A antibodies. Neutralizing antibody responses to reproductive hormones can be used for contraception. Neutralizing antibody response to hormones and other soluble factors that are required for the growth of particular tumors are additional vaccination targets.

Analogous methods as described above for the use of anti-NKG2A antibodies can be used for induction of therapeutic autoimmune responses to treat patients having an inappropriate accumulation of other self-antigens, such as amyloid deposits, including Aβ in Alzheimer's disease, cytokines such as TNF-α, and IgE.

Vaccines

In some aspects, anti-NKG2A antibodies described herein are used to stimulate antigen-specific immune responses administering an anti-NKG2A antibody along with an antigen of interest (e.g., a vaccine). Accordingly, provided herein are methods of enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) the antigen; and (ii) an anti-NKG2A antibody, or antigen-binding fragment thereof, such that an immune response to the antigen in the subject is enhanced. The antibody can be a human anti-human NKG2A antibody (such as any of the human anti-NKG2A antibodies described herein). In some embodiments, the anti-NKG2A antibody is a chimeric or humanized antibody. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen or an antigen from a pathogen. Non-limiting examples of such antigens include those discussed in the sections herein, such as the tumor antigens (or tumor vaccines) discussed herein, or antigens from the viruses, bacteria or other pathogens described herein.

In certain embodiments, a peptide or fusion protein comprising the epitope to which an anti-NKG2A antibody binds is used as a vaccine instead of, or in addition to, an anti-NKG2A antibody.

Suitable routes of administering the antibody compositions (e.g., human monoclonal antibodies, multi specific and bispecific molecules and immunoconjugates) described herein in vivo and in vitro are well known in the art and can be selected by those of ordinary skill in the art. For example, the antibody compositions can be administered by intravenous or subcutaneous routes. Suitable dosages of the composition used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.

As previously described, anti-NKG2A antibodies described herein can be co-administered with one or other more therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent. The anti-NKG2A antibody can be linked to the agent (as an immuno-complex) or can be administered separately from the agent. In the latter case (separate administration), the anti-NKG2A antibody can be administered before, after, or concurrently with the agent or can be coadministered with other known therapies, e.g., an anti-cancer therapy, e.g., chemotherapy and/or radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient. Cisplatin is intravenously administered as a 100 mg/ml dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/ml dose once every 21 days. Co-administration of anti-NKG2A antibodies, or antigen binding fragments thereof, described herein with chemotherapeutic agents provides two anti-cancer agents that operate via different mechanisms to yield a cytotoxic effect to human tumor cells. Such coadministration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells, which would render them unreactive with the antibody.

Also within the scope described herein are kits comprising the antibody compositions described herein (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. The kit can further contain at least one additional reagent, or one or more additional human anti-NKG2A antibodies described herein (e.g., a human antibody having a complementary activity which binds to an epitope in NKG2A antigen distinct from the first human antibody). Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

Combination Therapies

In one aspect, provided herein are methods of combination therapy, e.g., for the treatment of cancer, in which an anti-hNKG2A antibody is administered in connection with one or more additional agents, e.g., antibodies, that are effective in stimulating immune responses to thereby further enhance, stimulate or upregulate immune responses in a subject, including a human subject. Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an anti-hNKG2A antibody (e.g., NKG2A.9, NKG2A.11, and 13F3.A4) in conjunction with another anti-cancer agent or cancer therapy. In some embodiments, an anti-hNKG2A antibody may be administered in conjunction with a chemotherapy or chemotherapeutic agent or with a radiation therapy or radiotherapeutic agent, as described above. In some embodiments, an anti-hNKG2A antibody may be administered in conjunction with an agonist antibody, such as anti-hICOS antibody. In some embodiments, an anti-hNKG2A antibody may be administered in conjunction with a targeted therapy or targeted therapeutic agent. In some embodiments, an anti-hNKG2A antibody may be administered in conjunction with an immunotherapy or immunotherapeutic agent, for example a monoclonal antibody.

In some embodiments, an anti-hNKG2A antibody described herein can be combined with (i) an agonist of another co-stimulatory receptor and/or (ii) an antagonist of an inhibitory signal on T cells. In some embodiments, a combination therapy comprising an anti-hNKG2A antibody and the agonist and/or antagonist results in an enhanced antigen-specific T cell response in a subject. In some embodiment, anti-hNKG2A antibodies described herein may be administered in conjunction with an agent that targets a co-stimulatory and co-inhibitory molecules that is a member of the immunoglobulin super family (IgSF) to increase an immune response. In some embodiment, anti-hNKG2A antibodies described herein may be administered in conjunction with an agent that targets a ligand of a co-stimulatory or co-inhibitory molecule. A family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane bound ligands that bind to co-stimulatory or co-inhibitory receptors is the TNF family of molecules that bind to cognate TNF receptor family members, which include CD40, CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137/4-1BB, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DRS, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, Lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, Lymphotoxin α 1β2, FAS, FASL, RELT, DR6, TROY, NGFR.

In another aspect, anti-hNKG2A antibodies can be used in combination with antagonists of cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF; or other “immunosuppressive cytokines,” or cytokines that stimulate T cell activation, for stimulating an immune response, e.g., for treating proliferative diseases, such as cancer.

In one aspect, T cell responses are stimulated by a combination of an anti-hNKG2A antibody described herein and one or more of (i) an antagonist of a protein that inhibits T cell activation (e.g., immune checkpoint inhibitors) such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, and (ii) an agonist of a protein that stimulates T cell activation such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and may be combined with anti-hNKG2A, e.g., those described herein, for treating cancer, include: YERVOY®/ipilimumab or tremelimumab (to CTLA-4), galiximab (to B7.1), BMS-936558 (to PD-1), pidilizumab/CT-011 (to PD-1), KEYTRUDA®/pembrolizumab/MK-3475 (to PD-1), AMP224 (to B7-DC/PD-L2), BMS-936559 (to B7-H1), MPDL3280A (to B7-H1), CP-870893 or dacetuzumab/SGN-40 (CD40-Kirkwood et al. (2012) CA Cancer J. Clin. 62:309; Vanderheide & Glennie (2013) Clin. Cancer Res. 19:1035), AMG557 (to B7H2), MGA271 (to B7H3—WO 11/109400), IMP321 (to LAG-3), urelumab/BMS-663513 and PF-05082566 (to CD137/4-1BB), varlilumab/CDX-1127 (to CD27), MEDI-6383 and MEDI-6469 (to OX40), RG-7888 (to OX40L—WO 06/029879), Atacicept (to TACI), muromonab-CD3 (to CD3), ipilumumab (to CTLA-4). Accordingly, in one embodiment an anti-hNKG2A antibody (such as NKG2A.9) is combined with an anti-PD-1 antibody (such as nivolumab) and/or an anti-CTLA-4 antibody (such as ipilimumab).

Other molecules that can be combined with anti-hNKG2A antibodies for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, anti-hNKG2A antibodies can be combined with antagonists of KIR (e.g., lirilumab).

Yet other agents for combination therapies include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1R antagonists such as CSF-1R antagonist antibodies including RG7155 (WO 11/70024, WO 11/107553, WO 11/131407, WO 13/87699, WO 13/119716, WO 13/132044) or FPA-008 (WO 11/140249; WO 13/169264; WO 14/036357).

In some embodiments, anti-hNKG2A antibodies described herein are used together with one or more of agonistic agents that ligate positive co-stimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, and one or more agents that increase systemically the frequency of anti-tumor T cells, agents that overcome distinct immune suppressive pathways within the tumor microenvironment (e.g., block inhibitory receptor engagement (e.g., PD-L1/PD-1 interactions), deplete or inhibit Tregs (e.g., using an anti-CD25 monoclonal antibody (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibit metabolic enzymes such as IDO, or reverse/prevent T cell anergy or exhaustion) and agents that trigger innate immune activation and/or inflammation at tumor sites.

Provided herein are methods for stimulating an immune response in a subject comprising administering to the subject an anti-hNKG2A antibody and one or more additional immunostimulatory antibodies, such as a PD-1 antagonist, e.g., antagonist antibody, a PD-L1 antagonist, e.g., antagonist antibody, a CTLA-4 antagonist, e.g., antagonist antibody and/or a LAG3 antagonist, e.g., an antagonist antibody, such that an immune response is stimulated in the subject, for example to inhibit tumor growth or to stimulate an anti-viral response. In one embodiment, the subject is administered an anti-hNKG2A antibody and an antagonist anti-PD-1 antibody. In one embodiment, the subject is administered an anti-hNKG2A antibody and an antagonist anti-PD-L1 antibody. In one embodiment, the subject is administered an anti-hNKG2A antibody and an antagonist anti-CTLA-4 antibody. In one embodiment, the at least one additional immunostimulatory antibody (e.g., an antagonist anti-PD-1, an antagonist anti-PD-L1, an antagonist anti-CTLA-4 and/or an antagonist anti-LAG3 antibody) is a human antibody. Alternatively, the at least one additional immunostimulatory antibody can be, for example, a chimeric or humanized antibody (e.g., prepared from a mouse or hamster anti-PD-1, anti-PD-L1, anti-CTLA-4 and/or anti-LAG3 antibody).

Provided herein are methods for treating a hyperproliferative disease (e.g., cancer), comprising administering an anti-hNKG2A antibody and an antagonist PD-1 antibody to a subject. In some embodiments, the cancer is non-small cell lung cancer (NSCLC) or colorectal cancer (CRC). In some embodiments, the cancer is characterized by tumors with (i) elevated levels of HLA-E; and/or (ii) higher tumor mutation burden. In certain embodiments, the anti-hNKG2A antibody is administered at a subtherapeutic dose, the anti-PD-1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Also provided herein are methods for altering an adverse event associated with treatment of a hyperproliferative disease with an immunostimulatory agent. In one embodiment, the method comprises administering an anti-hNKG2A antibody and a subtherapeutic dose of anti-PD-1 antibody to a subject. In some embodiments, the subject is a human. In some embodiments, the anti-PD-1 antibody is a human monoclonal antibody.

In some embodiments, anti-PD-1 antibodies that are known in the art are used in the presently described methods in combniatinon with the anti-NKG2A antibodies described herein. Various human monoclonal antibodies that bind specifically to PD-1 with high affinity have been disclosed in U.S. Pat. No. 8,008,449. Anti-PD-1 human antibodies disclosed in U.S. Pat. No. 8,008,449 have been demonstrated to exhibit one or more of the following characteristics: (a) bind to human PD-1 with a K_(D) of 1×10⁻⁷ M or less, as determined by surface plasmon resonance using a Biacore biosensor system; (b) do not substantially bind to human CD28, CTLA-4 or ICOS; (c) increase T-cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay; (d) increase interferon-γ production in an MLR assay; (e) increase IL-2 secretion in an MLR assay; (f) bind to human PD-1 and cynomolgus monkey PD-1; (g) inhibit the binding of PD-L1 and/or PD-L2 to PD-1; (h) stimulate antigen-specific memory responses; (i) stimulate antibody responses; and (j) inhibit tumor cell growth in vivo. Anti-PD-1 antibodies usable in the present invention include monoclonal antibodies that bind specifically to human PD-1 and exhibit at least one, in some embodiments, at least five, of the preceding characteristics.

Other anti-PD-1 monoclonal antibodies have been described in, for example, U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757 and 8,354,509, US Publication No. 2016/0272708, and PCT Publication Nos. WO 2012/145493, WO 2008/156712, WO 2015/112900, WO 2012/145493, WO 2015/112800, WO 2014/206107, WO 2015/35606, WO 2015/085847, WO 2014/179664, WO 2017/020291, WO 2017/020858, WO 2016/197367, WO 2017/024515, WO 2017/025051, WO 2017/123557, WO 2016/106159, WO 2014/194302, WO 2017/040790, WO 2017/133540, WO 2017/132827, WO 2017/024465, WO 2017/025016, WO 2017/106061, WO 2017/19846, WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540 each of which is incorporated by reference in its entirety.

In some embodiments, the anti-PD-1 antibody is nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO2008/156712), PDR001 (Novartis; see WO 2015/112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012/145493), cemiplimab (Regeneron; also known as REGN-2810; see WO 2015/112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; see WO 2015/35606 and US 2015/0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015/085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014/179664), GLS-010 (Wuxi/Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014/194302), AGEN2034 (Agenus; see WO 2017/040790), MGA012 (Macrogenics, see WO 2017/19846), or IBI308 (Innovent; see WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540).

In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the down-regulation of antitumor T-cell functions (U.S. Pat. No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody directed against human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Pat. Nos. 8,354,509 and 8,900,587.

Anti-PD-1 antibodies usable in the disclosed methods also include isolated antibodies that bind specifically to human PD-1 and cross-compete for binding to human PD-1 with any anti-PD-1 antibody disclosed herein, e.g., nivolumab (see, e.g., U.S. Pat. Nos. 8,008,449 and 8,779,105; WO 2013/173223). In some embodiments, the anti-PD-1 antibody binds the same epitope as any of the anti-PD-1 antibodies described herein, e.g., nivolumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody, e.g., nivolumab, by virtue of their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO 2013/173223).

In certain embodiments, the antibodies that cross-compete for binding to human PD-1 with, or bind to the same epitope region of human PD-1 antibody, nivolumab, are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

Anti-PD-1 antibodies usable in the methods of the disclosed invention also include antigen-binding portions of the above antibodies. It has been amply demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

Anti-PD-1 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to PD-1 with high specificity and affinity, block the binding of PD-L1 and or PD-L2, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-1 “antibody” includes an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits the functional properties similar to those of whole antibodies in inhibiting ligand binding and up-regulating the immune system. In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1.

Provided herein are methods for treating a hyperproliferative disease (e.g., cancer), comprising administering an anti-hNKG2A antibody and an antagonist PD-L1 antibody to a subject. In certain embodiments, the anti-hNKG2A antibody is administered at a subtherapeutic dose, the anti-PD-L1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Provided herein are methods for altering an adverse event associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-NKG2A antibody and a subtherapeutic dose of anti-PD-L1 antibody to a subject. In certain embodiments, the subject is human. In certain embodiments, the anti-PD-L1 antibody is a human sequence monoclonal antibody and the anti-hNKG2A antibody is a humanized monoclonal antibody, such as an antibody comprising the CDRs or variable regions of the antibodies disclosed herein.

Anti-PD-L1 antibodies that are known in the art can be used in the methods of the present disclosure. Examples of anti-PD-L1 antibodies useful in the methods of the present disclosure include the antibodies disclosed in U.S. Pat. No. 9,580,507. Anti-PD-L1 human monoclonal antibodies disclosed in U.S. Pat. No. 9,580,507 have been demonstrated to exhibit one or more of the following characteristics: (a) bind to human PD-L1 with a K_(D) of 1×10-7 M or less, as determined by SPR using a Biacore biosensor system; (b) increase T-cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay; (c) increase interferon-γ production in an MLR assay; (d) increase IL-2 secretion in an MLR assay; (e) stimulate antibody responses; and (f) reverse the effect of T regulatory cells on T cell effector cells and/or dendritic cells. Anti-PD-L1 antibodies usable in the present invention include monoclonal antibodies that bind specifically to human PD-L1 and exhibit at least one, in some embodiments, at least five, of the preceding characteristics.

In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Pat. No. 7,943,743 and WO 2013/173223), atezolizumab (Roche; also known as TECENTRIQ®; MPDL3280A, RG7446; see U.S. Pat. No. 8,217,149; see, also, Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; also known as IMFINZI™, MEDI-4736; see WO 2011/066389), avelumab (Pfizer; also known as BAVENCIO®, MSB-0010718C; see WO 2013/079174), STI-1014 (Sorrento; see WO2013/181634), CX-072 (Cytomx; see WO2016/149201), KN035 (3D Med/Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017/034916), or CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR:Abstract 4606 (April 2016)).

In certain embodiments, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

In certain embodiments, the PD-L1 antibody is durvalumab (IMFINZI™). Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

In certain embodiments, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

In other embodiments, the anti-PD-L1 monoclonal antibody is 28-8, 28-1, 28-12, 29-8, 5H1, or any combination thereof.

Anti-PD-L1 antibodies usable in the disclosed methods also include isolated antibodies that bind specifically to human PD-L1 and cross-compete for binding to human PD-L1 with any anti-PD-L1 antibody disclosed herein, e.g., atezolizumab, durvalumab, and/or avelumab. In some embodiments, the anti-PD-L1 antibody binds the same epitope as any of the anti-PD-L1 antibodies described herein, e.g., atezolizumab, durvalumab, and/or avelumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody, e.g., atezolizumab and/or avelumab, by virtue of their binding to the same epitope region of PD-L1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and/or avelumab in standard PD-L1 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO 2013/173223).

In certain embodiments, the antibodies that cross-compete for binding to human PD-L1 with, or bind to the same epitope region of human PD-L1 antibody as, atezolizumab, durvalumab, and/or avelumab, are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

Anti-PD-L1 antibodies usable in the methods of the disclosed invention also include antigen-binding portions of the above antibodies. It has been amply demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

Anti-PD-L1 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to PD-L1 with high specificity and affinity, block the binding of PD-1, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-L1 “antibody” includes an antigen-binding portion or fragment that binds to PD-L1 and exhibits the functional properties similar to those of whole antibodies in inhibiting receptor binding and up-regulating the immune system. In certain embodiments, the anti-PD-L1 antibody or antigen-binding portion thereof cross-competes with atezolizumab, durvalumab, and/or avelumab for binding to human PD-L1.

In one embodiment, the anti-hNKG2A antibody of the present invention is combined with an antagonist of PD-1/PD-L1 signaling, such as a PD-1 antagonist (e.g., nivolumab, also known as MDX1106, as described in WO 06/121168) or a PD-L1 antagonist, in combination with a third immunotherapeutic agent (e.g., an anti-ICOS antibody, such as ICOS.33 IgG1f S267E (as described in U.S. Pat. No. 10,251,945), combined with nivolumab and ipilimumab). In one embodiment, the third immunotherapeutic agent is a CTLA-4 antagonist antibody. In certain embodiments, the anti-CTLA-4 antibody is YERVOY® (ipilimumab or antibody 10D1, described in PCT Publication WO 01/14424) or tremelimumab (formerly ticilimumab, CP-675,206). In one embodiment, the third immunotherapeutic agent is a GITR antagonist or an OX-40 antagonist, such as the anti-GITR or anti-OX40 antibodies disclosed herein. In one embodiment, the third immunotherapeutic agent is a GITR agonist, such as an agonistic GITR antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO06/105021, WO09/009116) and MK-4166 (WO11/028683). In one embodiment, the third immunotherapeutic agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod, or NLG-919 (WO09/73620, WO09/1156652, WO11/56652, WO12/142237).

Provided herein are methods for treating a hyperproliferative disease (e.g., cancer), comprising administering an anti-hNKG2A antibody described herein and a CTLA-4 antagonist antibody to a subject. In certain embodiments, the anti-hNKG2A antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Provided herein are methods for altering an adverse event associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-hNKG2A antibody and a subtherapeutic dose of anti-CTLA-4 antibody to a subject. In certain embodiments, the subject is human.

Anti-CTLA-4 antibodies that are known in the art can be used in the methods of the present disclosure. Anti-CTLA-4 antibodies of the instant invention bind to human CTLA-4 so as to disrupt the interaction of CTLA-4 with a human B7 receptor. Because the interaction of CTLA-4 with B7 transduces a signal leading to inactivation of T-cells bearing the CTLA-4 receptor, disruption of the interaction effectively induces, enhances or prolongs the activation of such T cells, thereby inducing, enhancing or prolonging an immune response.

Human monoclonal antibodies that bind specifically to CTLA-4 with high affinity have been disclosed in U.S. Pat. No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies have been described in, for example, U.S. Pat. Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121 and International Publication Nos. WO 2012/122444, WO 2007/113648, WO 2016/196237, and WO 2000/037504, each of which is incorporated by reference herein in its entirety. Anti-CTLA-4 antibodies useful for the present invention include monoclonal antibodies that bind specifically to human CTLA-4 and exhibit at least one, at least two, or at least three of the preceding characteristics.

In certain embodiments, the CTLA-4 antibody is ipilimumab (also known as YERVOY®, MDX-010, 10D1; see U.S. Pat. No. 6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; see WO 2016/196237), or tremelimumab (AstraZeneca; also known as ticilimumab, CP-675,206; see WO 2000/037504 and Ribas, Update Cancer Ther. 2(3): 133-39 (2007)). In particular embodiments, the anti-CTLA-4 antibody is ipilimumab.

In particular embodiments, the CTLA-4 antibody is ipilimumab for use in the methods disclosed herein. Ipilimumab is a fully human, IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligands, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma.

In particular embodiments, the CTLA-4 antibody is tremelimumab.

In particular embodiments, the CTLA-4 antibody is MK-1308.

In particular embodiments, the CTLA-4 antibody is AGEN-1884.

Anti-CTLA-4 antibodies usable in the disclosed methods also include isolated antibodies that bind specifically to human CTLA-4 and cross-compete for binding to human CTLA-4 with any anti-CTLA-4 antibody disclosed herein, e.g., ipilimumab and/or tremelimumab. In some embodiments, the anti-CTLA-4 antibody binds the same epitope as any of the anti-CTLA-4 antibodies described herein, e.g., ipilimumab and/or tremelimumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody, e.g., ipilimumab and/or tremelimumab, by virtue of their binding to the same epitope region of CTLA-4. Cross-competing antibodies can be readily identified based on their ability to cross-compete with ipilimumab and/or tremelimumab in standard CTLA-4 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO 2013/173223).

In certain embodiments, the antibodies that cross-compete for binding to human CTLA-4 with, or bind to the same epitope region of human CTLA-4 antibody as, ipilimumab and/or tremelimumab, are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

Anti-CTLA-4 antibodies usable in the methods of the disclosed invention also include antigen-binding portions of the above antibodies. It has been amply demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

Anti-CTLA-4 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to CTLA-4 with high specificity and affinity, block the activity of CTLA-4, and disrupt the interaction of CTLA-4 with a human B7 receptor. In any of the compositions or methods disclosed herein, an anti-CTLA-4 “antibody” includes an antigen-binding portion or fragment that binds to CTLA-4 and exhibits the functional properties similar to those of whole antibodies in inhibiting the interaction of CTLA-4 with a human B7 receptor and up-regulating the immune system. In certain embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof cross-competes with ipilimumab and/or tremelimumab for binding to human CTLA-4.

In one embodiment, the anti-hNKG2A antibody of the present invention is combined with an anti-CTLA-4 antibody, in combination with a third immunotherapeutic agent. In one embodiment the third immunotherapeutic agent is a GITR antagonist or an OX-40 antagonist, such as the anti-GITR or anti-OX40 antibodies disclosed herein. In one embodiment, the third immunotherapeutic agent is a GITR agonist, such as an agonistic GITR antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO06/105021, WO09/009116) and MK-4166 (WO11/028683). In one embodiment, the third immunotherapeutic agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod, or NLG-919 (WO09/73620, WO09/1156652, WO11/56652, WO12/142237).

Provided herein are methods for treating a hyperproliferative disease (e.g., cancer), comprising administering an anti-hNKG2A antibody and an anti-LAG-3 antibody to a subject. In further embodiments, the agonist anti-hNKG2A antibody is administered at a subtherapeutic dose, the anti-LAG-3 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Provided herein are methods for altering an adverse event associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-hNKG2A antibody and a subtherapeutic dose of anti-LAG-3 antibody to a subject. In certain embodiments, the subject is human. In certain embodiments, the anti-hNKG2A antibody is a human sequence monoclonal antibody and the anti-hNKG2A antibody is a humanized monoclonal antibody, such as an antibody comprising the CDRs or variable regions of the antibodies disclosed herein. Examples of anti-LAG3 antibodies include antibodies comprising the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2 or 17E5, which are described in U.S. Patent Publication No. US2011/0150892 and WO2014/008218. In one embodiment, an anti-LAG-3 antibody is BMS-986016. Other anti-LAG-3 antibodies that can be used include IMP731 described in US 2011/007023 or IMP-321. Anti-LAG-3 antibodies that compete with and/or bind to the same epitope as that of any of these antibodies may also be used in combination treatments.

In certain embodiments, the anti-LAG-3 antibody binds to human LAG-3 with a K_(D) of 5×10⁻⁸ M or less, binds to human LAG-3 with a K_(D) of 1×10⁻⁸M or less, binds to human LAG-3 with a K_(D) of 5×10⁻⁹M or less, or binds to human LAG-3 with a K_(D) of between 1×10⁻⁸M and 1×10⁻¹⁰ M or less.

Administration of anti-hNKG2A antibodies described herein and other antagonists, e.g., antagonist antibodies, to one or more second target antigens such as LAG-3 and/or CTLA-4 and/or PD-1 and/or PD-L1 enhances the immune response to cancerous cells in the patient. Cancers whose growth may be inhibited using the antibodies of the instant disclosure include cancers typically responsive to immunotherapy. Examples of cancers for treatment with the combination therapy described herein include, but are not limited to, the described above in the discussion of monotherapy with anti-hNKG2A antibodies.

In certain embodiments, the combination of therapeutic antibodies discussed herein can be administered concurrently as a single composition in a pharmaceutically acceptable carrier, or concurrently as separate compositions with each antibody in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies can be administered sequentially. For example, an anti-CTLA-4 antibody and an anti-hNKG2A antibody can be administered sequentially, such as anti-CTLA-4 antibody being administered first and anti-hNKG2A antibody second, or anti-hNKG2A antibody being administered first and anti-CTLA-4 antibody second. Additionally or alternatively, an anti-PD-1 antibody and an anti-hNKG2A antibody can be administered sequentially, such as anti-PD-1 antibody being administered first and anti-hNKG2A antibody second, or anti-hNKG2A antibody being administered first and anti-PD-1 antibody second. Additionally or alternatively, an anti-PD-L1 antibody and anti-hNKG2A antibody can be administered sequentially, such as anti-PD-L1 antibody being administered first and anti-hNKG2A antibody second, or anti-hNKG2A antibody being administered first and anti-PD-L1 antibody second. Additionally or alternatively, an anti-LAG-3 antibody and anti-hNKG2A antibody can be administered sequentially, such as anti-LAG-3 antibody being administered first and anti-hNKG2A antibody second, or anti-hNKG2A antibody being administered first and anti-LAG-3 antibody second.

Furthermore, if more than one dose of the combination therapy is administered sequentially, the order of the sequential administration can be reversed or kept in the same order at each time point of administration, sequential administrations can be combined with concurrent administrations, or any combination thereof. For example, the first administration of a combination anti-CTLA-4 antibody and anti-hNKG2A antibody can be concurrent, the second administration can be sequential with anti-CTLA-4 antibody first and anti-hNKG2A antibody second, and the third administration can be sequential with anti-hNKG2A antibody first and anti-CTLA-4 antibody second, etc. Additionally or alternatively, the first administration of a combination anti-PD-1 antibody and anti-hNKG2A antibody can be concurrent, the second administration can be sequential with anti-PD-1 antibody first and anti-hNKG2A antibody second, and the third administration can be sequential with anti-hNKG2A antibody first and anti-PD-1 antibody second, etc. Additionally or alternatively, the first administration of a combination anti-PD-L1 antibody and anti-hNKG2A antibody can be concurrent, the second administration can be sequential with anti-PD-L1 antibody first and anti-hNKG2A antibody second, and the third administration can be sequential with anti-hNKG2A antibody first and anti-PD-L1 antibody second, etc. Additionally or alternatively, the first administration of a combination anti-LAG-3 antibody and anti-hNKG2A antibody can be concurrent, the second administration can be sequential with anti-LAG-3 antibody first and anti-hNKG2A antibody second, and the third administration can be sequential with anti-hNKG2A antibody first and anti-LAG-3 antibody second, etc. Another representative dosing scheme can involve a first administration that is sequential with anti-hNKG2A first and anti-CTLA-4 antibody (and/or anti-PD-1 antibody and/or anti-PD-L1 antibody and/or anti-LAG-3 antibody) second, and subsequent administrations may be concurrent.

In one embodiment, an anti-hNKG2A antibody, as sole immunotherapeutic agent, or the combination of an anti-hNKG2A antibody and one or more additional immunotherapeutic antibodies (e.g., anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibody) may be further combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below). An ICOS agonist and one or more additional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade) can also be further combined with standard cancer treatments. For example, an ICOS agonist and one or more additional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade) may be combined with chemotherapeutic regimes. In one embodiment, an anti-hNKG2A antibody is administered to a patient with an anti-CTLA-4 antibody and/or anti-PD-1 antibody and/or anti-PD-L1 antibody and/or anti-LAG-3 antibody in combination with dacarbazine for the treatment of melanoma. In one embodiment, an anti-hNKG2A antibody is administered to a patient with an anti-CTLA-4 antibody and/or anti-PD-1 antibody and/or anti-PD-L1 antibody and/or anti-LAG-3 antibody in combination with interleukin-2 (IL-2) for the treatment of cancer, including melanoma. Other combination therapies that may result in synergy with a combined anti-hNKG2A antibody with or without and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 antagonism through cytotoxicity include radiation, surgery, or hormone deprivation. In another embodiment, angiogenesis inhibitors may be combined with an anti-hNKG2A antibody and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 antagonism.

In one embodiment, an anti-hNKG2A antibody as sole immunotherapeutic agent, or a combination of an anti-hNKG2A antibody and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocking antibodies can also be used in combination with bispecific antibodies that target Fcα or Fcγ receptor-expressing effector cells to tumor cells. See, e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243. Bispecific antibodies can be used to target two separate antigens.

In one embodiment an anti-hNKG2A antibody as sole immunotherapeutic agent or a combination of an anti-hNKG2A antibody and additional immunostimulating agent, e.g., anti-CTLA-4 antibody and/or anti-PD-1 antibody and/or anti-PD-L1 antibody and/or anti-LAG-3 antibody, can be used in conjunction with an anti-neoplastic agent, such as RITUXAN® (rituximab), HERCEPTIN® (trastuzumab), BEXXAR® (tositumomab), ZEVALIN® (ibritumomab), CAMPATH® (alemtuzumab), LYMPHOCIDE® (eprtuzumab), AVASTIN (bevacizumab), and TARCEVA (erlotinib). By way of example and not wishing to be bound by theory, treatment with an anti-cancer antibody or an anti-cancer antibody conjugated to a toxin can lead to cancer cell death (e.g., tumor cells) which may potentiate an immune response mediated by the immunostimulating agent, e.g., anti-NKG2A antibody, anti-TIGIT antibody, anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-ICOS antibody, and/or anti-LAG-3 antibody. In one embodiment, a treatment of a hyperproliferative disease (e.g., a cancer tumor) can include an anti-cancer agent, e.g., antibody, in combination with an anti-NKG2A antibody and optionally an additional immunostimulating agent, e.g., anti-CTLA-4 antibody and/or anti-PD-1 antibody and/or anti-PD-L1 antibody and/or anti-LAG-3 antibody, concurrently or sequentially or any combination thereof, which can potentiate an anti-tumor immune responses by the host.

Provided herein are methods for reducing, ameliorating or abrogating an adverse event associated with treatment of a hyperproliferative disease (e.g., cancer) with an immunostimulatory agent, comprising administering an anti-NKG2A antibody with or without an anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibody, to a subject. In one embodiment, the method reduces the incidence of immunostimulatory therapeutic antibody-induced colitis or diarrhea by administering a non-absorbable steroid to the patient. As used herein, a “non-absorbable steroid” is a glucocorticoid that exhibits extensive first pass metabolism such that, following metabolism in the liver, the bioavailability of the steroid is low, i.e., less than about 20%. In one embodiment described herein, the non-absorbable steroid is budesonide. Budesonide is a locally-acting glucocorticosteroid, which is extensively metabolized, primarily by the liver, following oral administration. ENTOCORT EC® (Astra-Zeneca) is a pH- and time-dependent oral formulation of budesonide developed to optimize drug delivery to the ileum and throughout the colon. ENTOCORT EC® is approved in the U.S. for the treatment of mild to moderate Crohn's disease involving the ileum and/or ascending colon. The usual oral dosage of ENTOCORT EC® for the treatment of Crohn's disease is 6 to 9 mg/day. ENTOCORT EC® is released in the intestines before being absorbed and retained in the gut mucosa. Once it passes through the gut mucosa target tissue, ENTOCORT EC® is extensively metabolized by the cytochrome P450 system in the liver to metabolites with negligible glucocorticoid activity. Therefore, the bioavailability is low (about 10%). The low bioavailability of budesonide results in an improved therapeutic ratio compared to other glucocorticoids with less extensive first-pass metabolism. Budesonide results in fewer adverse effects, including less hypothalamic-pituitary suppression, than systemically-acting corticosteroids. However, chronic administration of ENTOCORT EC® can result in systemic glucocorticoid effects such as hypercorticism and adrenal suppression. See PDR 58^(th) ed. 2004; 608-610.

In one embodiment, an anti-NKG2A antibody with or without CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 antagonist in conjunction with a non-absorbable steroid can be further combined with a salicylate. Salicylates include 5-ASA agents such as, for example: sulfasalazine (AZULFIDINE®, Pharmacia & UpJohn); olsalazine (DIPENTUM®, Pharmacia & UpJohn); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.); and mesalamine (ASACOL®, Procter & Gamble Pharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay).

In accordance with the methods described herein, a salicylate administered in combination with an anti-NKG2A antibody with or without anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or LAG-3 antibodies and a non-absorbable steroid includes any overlapping or sequential administration of the salicylate and the non-absorbable steroid for the purpose of decreasing the incidence of colitis induced by the immunostimulatory antibodies. Thus, for example, methods for reducing the incidence of colitis induced by the immunostimulatory antibodies described herein encompass administering a salicylate and a non-absorbable concurrently or sequentially (e.g., a salicylate is administered 6 hours after a non-absorbable steroid), or any combination thereof. Further, a salicylate and a non-absorbable steroid can be administered by the same route (e.g., both are administered orally) or by different routes (e.g., a salicylate is administered orally and a non-absorbable steroid is administered rectally), which may differ from the route(s) used to administer the anti-NKG2A antibody and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies.

The anti-NKG2A antibody antibodies and combination antibody therapies described herein may also be used in conjunction with other well-known therapies that are selected for their particular usefulness against the indication being treated (e.g., cancer). Combinations of the anti-NKG2A antibody antibodies described herein may be used sequentially with known pharmaceutically acceptable agent(s).

In one embodiment, the anti-NKG2A antibody antibodies and combination antibody therapies described herein can be used in combination (e.g., simultaneously or separately) with an additional treatment, such as irradiation, chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-fu, or camptothecin+apo21/TRAIL (a 6× combo)), one or more proteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3I-2′ (bcl-xl inhibitor), indoleamine dioxygenase-1 (IDO1) inhibitor (e.g., INCB24360), AT-101 (R-(−) -gossypol derivative), ABT-263 (small molecule), GX-15-070 (obatoclax), or MCL-1 (myeloid leukemia cell differentiation protein-1) antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g., smac7, smac4, small molecule smac mimetic, synthetic smac peptides (see Fulda et al., Nat Med 2002; 8:808-15), ISIS23722 (LY2181308), or AEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab), angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g., AVASTIN®), synthetic triterpenoids (see Hyer et al., Cancer Research 2005; 65:4799-808), c-FLIP (cellular FLICE-inhibitory protein) modulators (e.g., natural and synthetic ligands of PPARγ (peroxisome proliferator-activated receptor γ), 5809354 or 5569100), kinase inhibitors (e.g., Sorafenib), trastuzumab, cetuximab, Temsirolimus, mTOR inhibitors such as rapamycin and temsirolimus, Bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, Lenalildomide, GSK3β inhibitors, IAP inhibitors and/or genotoxic drugs.

The anti-NKG2A antibody antibodies and combination antibody therapies described herein can further be used in combination with one or more anti-proliferative cytotoxic agents. Classes of compounds that may be used as anti-proliferative cytotoxic agents include, but are not limited to, the following:

Alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN™) fosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for combining with anti-NKG2A antibody antibodies, without limitation, taxanes, paclitaxel (paclitaxel is commercially available as TAXOL′), docetaxel, discodermolide (DDM), dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D, desoxyepothilone B1, [17]-dehydrodesoxyepothilone B, [18] dehydrodesoxyepothilones B, C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A, trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone B10, discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (Discodermolide), TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389), Hemiasterlin (HTI-286), E-7974, Cyrptohycins, LY-355703, Maytansinoid immunoconjugates (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin, 17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, and cryptothilone 1, in addition to other microtubuline stabilizing agents known in the art.

In some embodiments it may be desirable to render aberrantly proliferative cells quiescent in conjunction with or prior to treatment with anti-NKG2A antibody antibodies described herein, e.g., by administering to the patient hormones and steroids (including synthetic analogs), such as 17a-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, ZOLADEX™. When employing the methods or compositions described herein, other agents used in the modulation of tumor growth or metastasis in a clinical setting, such as antimimetics, can also be administered as desired.

Methods for the safe and effective administration of chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, N.J. 07645-1742, USA); the disclosure of which is incorporated herein by reference thereto.

The chemotherapeutic agent(s) and/or radiation therapy can be administered according to therapeutic protocols known in the art. It will be apparent to those skilled in the art that the administration of the chemotherapeutic agent(s) and/or radiation therapy can be varied depending on the disease being treated and the known effects of the chemotherapeutic agent(s) and/or radiation therapy on that disease. Also, in accordance with the knowledge of the skilled clinician, the therapeutic protocols (e.g., dosage amounts and times of administration) can be varied in view of the observed effects of the administered therapeutic agents on the patient, and in view of the observed responses of the disease to the administered therapeutic agents.

Outcomes

Tumor response is determined, for example, by modified Response Evaluation Criteria in Solid Tumors (RECIST) established by the NCI.

With respect to target lesions, responses to therapy may include:

Complete Response (CR) Disappearance of all target lesions. Any (RECIST V1.1) pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. Partial Response (PR) At least a 30% decrease in the sum of the (RECIST V1.1) diameters of target lesions, taking as reference the baseline sum diameters. Progressive Disease (PD) At least a 20% increase in the sum of the (RECIST V1.1) diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression). Stable Disease (SD) Neither sufficient shrinkage to qualify for (RECIST V1.1) PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study. Immune-related Complete Response Disappearance of all target lesions. Any (irCR) pathological lymph nodes (whether target (irRECIST) or non-target) must have reduction in short axis to <10 mm. Immune-related Partial Response (irPR) At least a 30% decrease in the sum of (irRECIST) diameters of target lesions and all new measurable lesions (e.g., Percentage Change in Tumor Burden), taking as reference the baseline sum diameters. Note: the appearance of new measurable lesions is factored into the overall Tumor Burden, but does not automatically qualify as progressive disease until the sum of the diameters increases by ≥20% when compared to nadir. Immune-related Progressive Disease At least a 20% increase in Tumor Burden (irPD) (e.g., the sum of diameters of target (irRECIST) lesions, and any new measurable lesions) taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. Tumor assessments using immune-related criteria for progressive disease incorporates the contribution of new measurable lesions. Each net percentage change in tumor burden per assessment accounts for the size and growth kinetics of both old and new lesions as they appear. Immune-related Stable Disease (irSD) Neither sufficient shrinkage to qualify for (irRECIST) irPR nor sufficient increase to qualify for irPD, taking as reference the smallest sum diameters while on study.

With respect to non-target lesions, responses to therapy may include:

Complete Response (CR) Disappearance of all non-target lesions. (RECIST V1.1) All lymph nodes must be non-pathological in size (<10 mm short axis). Non-CR/Non-PD Persistence of one or more non-target (RECIST V1.1) lesion(s). Progressive Disease (PD) Unequivocal progression of existing non- (RECIST V1.1) target lesions. The appearance of one or more new lesions is also considered progression. Immune-related Complete Response Disappearance of all non-target lesions. (irCR) All lymph nodes must be non-pathological (irRECIST) in size (<10 mm short axis). Immune-related Progressive Disease Increases in number or size of non-target (irPD) lesion(s) does not constitute progressive (irRECIST) disease unless/until Tumor Burden increases by 20% (e.g., the sum of the diameters at nadir of target lesions and any new measurable lesions increases by the required amount). Non-target lesions are not considered in the definition of Stable Disease and Partial Response.

Patients treated according to the methods disclosed herein preferably experience improvement in at least one sign of cancer. In one embodiment, improvement is measured by a reduction in the quantity and/or size of measurable tumor lesions. In another embodiment, lesions can be measured on chest x-rays or CT or Mill films. In another embodiment, cytology or histology can be used to evaluate responsiveness to a therapy.

In one embodiment, the patient treated exhibits a complete response (CR), a partial response (PR), stable disease (SD), immune-related complete disease (irCR), immune-related partial response (irPR), or immune-related stable disease (irSD). In another embodiment, the patient treated experiences tumor shrinkage and/or decrease in growth rate, i.e., suppression of tumor growth. In another embodiment, unwanted cell proliferation is reduced or inhibited. In yet another embodiment, one or more of the following can occur: the number of cancer cells can be reduced; tumor size can be reduced; cancer cell infiltration into peripheral organs can be inhibited, retarded, slowed, or stopped; tumor metastasis can be slowed or inhibited; tumor growth can be inhibited; recurrence of tumor can be prevented or delayed; one or more of the symptoms associated with cancer can be relieved to some extent.

In other embodiments, administration of effective amounts of the anti-NKG2A antibody (or combinations anti-NKG2A antibody and at least one additional antibody, e.g., an anti-PD-1 antibody or anti-CTLA-4 antibody) according to any of the methods provided herein produces a reduction in size of a tumor, reduction in number of metastatic lesions appearing over time, complete remission, partial remission, or stable disease. In still other embodiments, the methods of treatment produce a comparable clinical benefit rate (CBR=CR+PR+SD≥6 months) better than that achieved by an anti-NKG2A antibody alone (or any one of the combined antibodies alone). In other embodiments, the improvement of clinical benefit rate is about 20% about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more compared to the anti-NKG2A antibody alone (or any one of the combined antibodies alone).

Vaccine Adjuvants

Anti-NKG2A antibodies described herein can be used to enhance antigen-specific immune responses by co-administration of an anti-NKG2A antibody with an antigen of interest, e.g., a vaccine. Accordingly, provided herein are methods of enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) the antigen; and (ii) an anti-NKG2A antibody, or antigen-binding fragment thereof, such that an immune response to the antigen in the subject is enhanced. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen or an antigen from a pathogen. Non-limiting examples of such antigens include those discussed in the sections above, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from the viruses, bacteria or other pathogens described above.

Detection and Diagnostics

In another aspect, provided herein are methods for detecting the presence of human NKG2A antigen in a sample, or measuring the amount of human NKG2A antigen, comprising contacting the sample, and a control sample, with an anti-NKG2A antibody, e.g., a monoclonal anti-human NKG2A antibody, or an antigen binding fragment thereof, that specifically binds to human NKG2A, under conditions that allow for formation of a complex between the antibody or fragment thereof and human NKG2A. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative the presence of human NKG2A antigen in the sample. Moreover, the anti-NKG2A antibodies described herein can be used to purify human NKG2A via immunoaffinity purification.

The present disclosure is further illustrated by the following examples, which should not be construed as limiting. The contents of all figures and all references, GenBank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLES

The following are non-limiting examples of anti-NKG2A antibodies, compositions, and methods of the invention. It is understood that various other embodiments may be practiced consistent with the general description provided herein.

Example 1 Generation of Anti-huNKG2A Antibodies

Fully human and chimeric anti-NKG2A monoclonal antibodies, and fully human and chimeric antibodies that bind to the same epitope and/or cross-block the binding of the anti-NKG2A antibodies are described in this disclosure. We desired antibodies that bound specifically to human and cynomolgus monkey NKG2A-expressing cells, but not to human and cynomolgus monkey NKG2C-expressing cells. The anti-NKG2A antibodies described herein may be generated using several methods as described in this example.

Generation, Screening, and Selection Methods of Anti-NKG2A Antibodies

1. Hybridoma Method and Development

Using a hybridoma method, anti-NKG2A antibodies were generated using transgenic mice that express human antibody genes. Human IgG transgenic Kunming (KM) mice were immunized through footpad injection with the plasma membrane of BAF3 (a murine pro-B cell line)-human NKG2A (BAF3-hNKG2A) and hCD94 cells or hNKG2A-hCD94-mFc protein. Lymph nodes from these mice were harvested, and hybridomas were generated using a CytoPulse electroporation instrument, which uses a pulse of electricity to increase permeability of the cell membrane, allowing, for example, DNA to be introduced into the cell. Antibodies specific for human NKG2A were selected in a primary screening by Fluorescent Microvolume Assay Technology (FMAT) for their ability to bind to CHO-S-human NKG2A. A secondary confirmation screen by Fluorescence-Activated Cell Sorting (FACS) analysis was performed to identify monoclonal antibodies that bind specifically to human NKG2A expressed on the cell surface of the CHO-S transfectant cells.

As shown in FIG. 1A, in step 101, 40 fusions of the lymph node cells fused to a myeloma cell line yielded 153 antibody clones.

Subsequently, in step 102, the hybridoma supernatants were tested for their ability to block binding of the HLA-E pentamer (a fluorescent labeled MHC-peptide complex, ProImmune) to human NKG2A-expressing cells. This assay identified antibodies that were able to block binding of the HLA-E pentamer to human NKG2A-expressing cells, and narrowed the number of antibody clones for further development from 153 to 28 antibody clones.

Next, antigen specificity was determined by the binding of purified antibodies to human or cynomolgus monkey NKG2A-expressing or NKG2C-expressing cells. Specifically, in Step 103, the 28 antibody clones that blocked the HLA-E/NKG2A interaction were further screened by the binding of purified antibodies to human NKG2A-expressing and screened against the binding to hCD94 or hNKG2C-expressing cells. In this step, three antibody clones (13F3.A4, 11H9.A4, and 4G5.D1) were discovered and selected for further investigation.

In Step 104, the three antibody clones (13F3.A4, 11H9.A4, and 4G5.D1) were screened for cross-reactivity to cynomolgus monkey NKG2A-expressing Chinese Hamster Ovary (CHO) cells.

In Step 105, various functional assays and epitope binning experiments described in the Examples herein were used to characterize the anti-NKG2A antibodies

In Step 106, further antibody optimization and mutational scan analysis, allowed for the discovery of additional anti-NKG2A antibodies with desired functional properties as described herein.

2. Antibody Library Generation Method

In addition to the hybridoma method to generate antibodies, the antibody library method was also used to generate additional anti-NKG2A antibodies. This method generated anti-NKG2A antibodies including the P1-069366 antibody, which showed desirable specific binding to both human and cynomolgus monkey NKG2A expressing cell lines, while binding to human NKG2C-expressing cells was advantageously not observed.

In this antibody library generation method, as shown in FIG. 1B, in step 107, nine transgenic mice expressing human antibodies were immunized with recombinant hNKG2A-hCD94-mFc fusion protein and plasma membranes from a cell line expressing cynomolgus monkey NKG2A. In step 108, the lymph nodes of the immunized animals were harvested and used to generate an immune antibody library. This library was expressed by yeast surface display and sorted using fluorescence-activated cell sorting (FACS) for antibodies that bound hNKG2A-hCD94-mFc recombinant protein, but not hNKG2C-hCD94-mFc recombinant protein. In step 109, the resulting population was sequenced using next generation sequencing (NGS), and antibodies of interest were synthesized and tested in assays. In step 110, functional characterization was performed. For example, to characterize the P1-069366 antibody, we measured binding of the antibody to CHO cell lines engineered to express human NKG2A-CD94, cynomolgus monkey NKG2A, or human NKG2C-CD94 by FACS. In addition, epitope binning was performed against, for example, the 13F3.A4 antibody and variants, including the NKG2A.9 antibody, as described in the Examples herein.

3. Single B Cell Cloning (SBCC) Method

Along with the hybridoma and antibody library methods to generate anti-NKG2A antibodies, a third method was used to generate over 200 additional anti-NKG2A antibodies.

Anti-huNKG2A antibodies were generated using transgenic mice that express human antibody genes. This SBCC method was used to generate over 200 human anti-NKG2A monoclonal antibodies. As shown in FIG. 1C, in step 111, human IgG transgenic (KM) mice were immunized through footpad injection with the plasma membranes of BAF3 cell lines expressing-human NKG2A and cynomolgus monkey NKG2A, respectively, and hNKG2A-hCD94-mFc protein. In step 112, lymph nodes from these mice were harvested and cells derived were used for the SBCC method. In step 113, 10×10⁶ lymph node cells were isolated from the lymph nodes. In step 114, the antigen specific B cell selection/sorting was based on cell surface human IgG expression, and we primarily used flow cytometry for sorting antigen positive (Ag+) B cells. The lymph nodes of the immunized animals were harvested and used to isolate antigen-specific B cells, that is, B cells specific to NKG2A.

Our FACS staining strategy required the use of soluble target antigen that is fluorescently labeled/biotinylated, and included a panel of B cell specific cell surface expressed markers, e.g. mouse CD19, mouse B220, and human IgG. This multiparametric FACS successfully resulted in the identification and sorting of NKG2A-specific B cells. Selective FACS staining resulted in isolation of hNKG2A-hCD94-mFc recombinant protein positive, but not hNKG2C-CD94-Fc recombinant protein B cells. The human IgG VH and VK genes from these sorted B cells were amplified by polymerase chain reaction (PCR) and molecularly expressed as recombinant human IgG1. The antibodies thus generated were tested in binding assays such as ELISA, HTRF and FACS.

The goal was to generate antibodies that specifically bound to NKG2A but not to NKG2C protein. We identified some antibodies that specifically bound to NKG2A, and some that were NKG2C negative. A very small number (rare) of B cell clones were identified that were cynomolgus and human NKG2A cross-reactive and NKG2C negative. NGS was performed on the SBCC derived B cell clones to generate human IgG VH and VK sequencing data. The selected panel of NKG2A specific antibodies were tested for further assays. However, none of the clones sufficiently blocked human NKG2A/HLA-E interaction, so these antibodies were not pursued for further development, as discussed in further detail in Example 3(2) below.

Example 2 Mutational Scan and Other Antibody Optimizations

A mutational scan was conducted in which a library of single-site mutations generated additional variants of anti-NKG2A antibodies, including the NKG2A.11 antibody (also known as 13F3.A4 VH 1107T, VK N30P).

FIG. 2 illustrates the mutational scan analysis. High throughput sequencing was combined with protein display to allow simultaneous measurement of the relative fitness of every possible single-site CDR mutant on a scale that would be onerous for a more traditional approach. Deep mutational scanning approaches have been described in Araya et al., Trends in Biotechnology 29: 435-442 (2001); Forsynth et al., mAbs 5:523-53 (2013); and Wrenbeck et al. (2017) Curr. Opin. Struct. Biol. 45:36.

First, in step 201, an antibody of interest was selected to generate mutations of the antibody. Specifically, the 13F3.A4 I107T anti-NKG2A antibody was selected. The binding paratope of the 13F3.A4 I107T hybridoma antibody to NGK2A was investigated using a deep mutational scan. Second, in step 202, an scFv (single chain) library was created where each individual amino acid substitution in the light chain CDR1 region (LCDR1), light chain CDR3 region (LCDR3), and heavy chain CDR1-3 regions (HCDR1, HCDR2, and HCDR3) was generated using NNK oligonucleotides. This library of single mutants was sequenced using next generation sequencing (NGS) techniques. In generating this library of single mutants, for each CDR sequence, multiple oligonucleotides were designed that individually incorporated an NNK codon at each position, where N=A, C, G, T, and K=G, T. The use of these degenerate codons allowed encoding of all 20 naturally-occurring amino acids (plus a stop codon) at the position where the NNK codon was incorporated. The Kabat definition was used for all CDRs except HCDR1, where the AbM definition was used (Abhinandan and Martin (2008) Mol. Immunol. 45:3832; Swindells et al. (2017)J Mol. Biol. 429:356), and position 102 of HCDR3 was not included in the analysis. Third, in step 203, using mRNA display (Xu L et al. (2002) Chemistry & Biology 9: 933; Roberts R W and J W Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297; Kurz et al. (2000) Nucleic Acids Res. 28(18): E83), the DNA library was taken through a single round of in vitro transcription and translation, during which the encoding mRNA was fused to its own scFv protein molecule via a puromycin linkage. During selection, any scFvs that bound biotin-labeled hNKG2A-hCD94-mFc fusion protein were captured by magnetic streptavidin beads, eluted, and amplified by PCR (step 205). As shown in step 204, antibodies that did not bind to human NKG2A were not selected for further testing and development. Finally, in step 206, the initial library generated in step 202 and the eluted DNA from step 205 were sequenced using next generation sequencing (NGS).

Next-Generation Sequencing (NGS) Data Analysis: During NGS data analysis, paired-end forward and reverse read sequences from NGS were assembled using FLASH (Magoc and Salzberg (2011) Bioinformatics 27:2957) and binned according to population, mutation position, and identity of the mutated amino acid. All sequences of poor quality and those containing multiple mutation sites were eliminated from the analysis. Next, the frequency of each sequence in the post-selection population was divided by the frequency in the starting population to derive an enrichment ratio (ER). In other words, the enrichment ratio is the counts of a particular sequence variant in the NKG2A-bound sample (step 205) divided by the counts in the initial library (step 202). This was then normalized to the enrichment ratio of the parental 13F3.A4 I107T antibody:

$\text{Enrichment Ratio} = {\frac{\text{Frequency after selection (step 205)}}{\text{Frequency in starting library (step 202)}}->\text{Normalize to parental value at each position}}$

In this manner, the effect on NKG2A binding of every single amino acid substitution in the CDR regions as discussed herein was assessed. FIG. 3 is an exemplary heat map that was generated using the mutational scan data analysis, and allows for the interpretation of the sequence-activity relationship of single amino acid substitutions. The HCDR1 sequence (AbM definition) of the parental clone 501 is shown, and the position in the sequence 502 is also shown. Generally, the error in this method is approximately two-fold. Thus, enrichment ratio (ER) values from 0.5 to 2 are considered to be neutral substitutions, i.e., substitutions that maintain binding properties to NKG2A protein. ER values greater than 2 are considered favorable or preferred for binding, and ER values less than 0.5 are considered unfavorable for binding. As an example, a substitution from parental clone N in position 35 to S results in an ER value of 5.16, which is greater than 2 and is, thus, an example of a favorable or preferred substitution.

The CDR positions analyzed using the mutational scan method are shown in FIG. 4. As discussed herein, the Kabat definition was used for all CDRs except HCDR1, where the AbM definition was used. To develop an anti-NKG2A antibody with desirable biophysical properties, the mutational scan data was used to remove the “NS” deamidation chemical liability in LCDR1 by identifying amino acid substitutions that would retain similar binding to NKG2A protein as the 13F3.A4 I107T antibody. Based on this analysis, as shown in FIG. 5A, N3OP in LCDR1 was chosen as a preferred (favorable) substitution, and the NKG2A.11 antibody (also known as 13F3 VH 1107T VK N30P) was generated. This molecule was synthesized and cloned into an IgG expression vector with the human IgG1.3f Fc region and human constant kappa (light chain). The IgG protein was expressed for further downstream characterization.

The mutation scan analysis provided a rich set of information about the effect of single amino acid substitutions in the 13F3.A4 I107T anti-NKG2A antibody on binding to NKG2A protein, as shown in FIGS. 5A-E and summarized in Table 1 below.

TABLE 1 Single site mutations in the CDR sequences of 13F3.A4 I107T anti-NKG2A antibody that maintain or increase the ability of the antibody to bind to NKG2A. Preferred Position substitutions that Amino acids that maintain (Under Rabat maintain or increase binding to NKG2A, including definition except binding to NKG2A parent amino acid sequence HCDR1 is under (heat maps are shown (heat maps are shown in CDR Region AbM definition) in FIG. 5A-E) FIG. 5A-E) LCDR1 24, X₁  G, A, C, S, T, V, L, I, M, K, R (parent), H, F, Y, W, N, D, E, Q 25, X₂  C, T, V, M P, G, A (parent), S, L, I, F, Y, N, E, Q 26, X₃  P, G, A, C, S(parent), T, V, L, I, M, K, R, H, F, Y, W, N, D, E, Q 27, X₄  G P, A, C, S, T, V, L, I, M, K, R, H, F, Y, W, N, D, E, Q (parent) 28, X₅  L P, A, C, S, T, V, I, M, K, R, H, F, Y, W, N, D, E, Q, G (parent) 29, X₆  C, H, F, N, D, P, G, A, S, T, V, L, I E (parent) M, K, R, Y, W, Q 30, X₇  P G, A, C, S, T, V, L, I, M, K, H, F, Y, W, N (parent), D, E, Q 31, X₈  P, G, C, D, E A, S (parent), T, L, I, M, H, F, Y, W, N, Q 32, X₉  V, I, F, Y, D, G, A (parent), C, S, E T, L, M, H, W, N 33, X₁₀ A, C, T, V, L (parent), I 34, X₁₁ E A (parent), T, V LCDR3 89, X₁₂ Q (parent) 90, X₁₃ D A, S, T, K, N, E, Q (parent) 91, X₁₄ C, M, H, W, N A, T, L, F (parent) 92, X₁₅ P, A, C, S, I, H, F, Y, W, N (parent), D, E, Q 93, X₁₆ V, D, E G, A, C, S (parent), T, L, I, M, K, R, H, F, Y, W, N, Q 94, X₁₇ M, H, F, Y (parent), W 95, X₁₈ P (parent), G, A, S, M, D, E, Q 96, X₁₉ C, L (parent), F, W 97, X₂₀ G, A, C, S, T (parent), V, I, M, K, H, F, Y, W, N, D, E, Q HCDR1 26, X₂₁ G (parent) 27, X₂₂ F (parent) 28, X₂₃ V G, A, C, S, T (parent), L, I, M, K, R, H, F, Y, N, D, E, Q 29, X₂₄ F (parent) 30, X₂₅ G, N P, A, C, S (parent), M, K, R, H, D, E, Q 31, X₂₆ G, A, V S (parent), T, E, Q 32, X₂₇ H (parent) 33, X₂₈ C A, S (parent), M, K, R 34, X₂₉ M (parent) 35, X₃₀ G, A, S T, V, N (parent), D HCDR2 50, X₃₁ S, Q G, A (parent), C, L, M, K, F, D 51, X₃₂ A S, V, L, I (parent), M, N, E, Q 52, X₃₃ S (parent) 52a, X₃₄  C S (parent) 53, X₃₅ Q S (parent) 54, X₃₆ S (parent) 55, X₃₇ F, Y, W A, C, S (parent), T, M, R, H 56, X₃₈ Y (parent) 57, X₃₉ I (parent) 58, X₄₀ G, A, S, M, K, R, H, F, Y (parent), N, E, Q 59, X₄₁ G, S, T, V, M, R, H, F, Y (parent), W, N, Q 60, X₄₂ H, Q P, G, A (parent), C, S, T, V, L, I, M, K, R, F, Y, W, N, D, E 61, X₄₃ P, G, A, C, S, T, V, L, I, M, K, R, H, F, Y, W, N, D (parent), E, Q 62, X₄₄ P, G, A, C, S (parent), T, V, L, I, M, K, R, H, F, Y, W, N, D, E, Q 63, X₄₅ P, G, A, C, S, T, V (parent), L, I, M, K, R, H, F, Y, W, N, D, E, Q 64, X₄₆ P, G, A, C, S, T, V, L, I, M, K (parent), R, H, F, Y, W, N, D, E, Q 65, X₄₇ P, G (parent), A, C, S, T, V, L, I, M, K, R, H, F, Y, W, N, D, E, Q HCDR3 95, X₄₈ E (parent) 96, X₄₉ M, E (parent) 97, X₅₀ W (parent) 98, X₅₁ G (parent) 99, X₅₂ S, L (parent), H 100, X₅₃  P (parent) 100a, X₅₄  F (parent) 101, X₅₅  D (parent) Using the full deep mutational scanning data, one of ordinary skill in the art would understand that many amino acid positions as shown in the data in FIG. 5A-E are tolerant to mutation, meaning that amino acid substitutions can be made at these positions and still maintain the desired functional ability to specifically bind to NKG2A protein. One of ordinary skill in the art would understand that certain positions, for example LCDR position 89 (as shown in FIG. 5B), as well as HCDR positions 26, 27, 29, 32, and 34 (as shown in FIG. 5C); HCDR positions 52, 52a, 53, 54, 56, 57 (as shown in FIG. 5D); and HDCR positions 95, 96, 97, 98, 99, 100, 100a, and 101 (as shown in FIG. 5E) were the most conserved, where only a single or a few amino acid types at these positions maintained binding to NKG2A protein. One of ordinary skill in the art would be able to design amino acid substitutions using this data to maintain binding to NKG2A protein.

Example 3

Discovery of Anti-NKG2A Antibodies with Desired, Unexpected Functional Characteristics after Analyzing Hundreds of Antibodies that Did not Exhibit Such Characteristics.

(1) Discovery, Design, and Experimental Testing of Anti-NKG2A Antibodies

Development of hundreds of anti-NKG2A antibodies was not pursued because the antibodies did not display certain desired functional properties as described herein. This difficulty in discovering anti-NKG2A antibodies with desired functional properties was partly due to the high sequence homology between the human NKG2A and human NKG2C proteins, as exemplified in FIG. 6. The sequence alignment was performed using Vector NTI software and the Align×program. FIG. 6 shows that 76% of amino acid residues (177 out of 233 amino acid residues) are conserved, and 6% of amino acid residues (13 of 233 amino acid residues) are similar between the canonical sequence of human NKG2A protein (SEQ ID NO: 182) and human NKG2C protein (hNKG2C, SEQ ID NO: 3).

It is well-known in the art that 20 different amino acids occur in nature, and that amino acids can be grouped together as in different classes and, thus, are “similar” based on their physicochemical properties. Amino acids that are physicochemically similar are often more interchangeable, that is, substitutable, than ones that are not. The following table provides a grouping of amino acids that have similar characteristics as defined, for example, by their side chains:

Amino acid characteristic Examples Polar Polar, positively Arginine Arg R charged Histidine His H Lysine Lys K Polar, negatively Aspartic acid Asp D charged Glumatic acid Glu E Polar, neutral Serine Ser S Threonine Thr T Asparagine Asn N Glutamine Gln Q Non-polar Non-polar aliphatic Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine Met M Non-polar aromatic Phenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp W Proline Pro P Glycine Cys C Special characteristics Asparagine/ Asx Aspartate Glutamine/ Glutamate Glx *Glycine does not have a side chain, and classification is not straight-forward. Glycine is generally found at the protein surface within loop or coil regions, which provides high flexibility to the polypeptide chain at these locations.

In summary, human NKG2A and human NKG2C share approximately 82% of the same or similar amino acid sequences. This high sequence homology between human NKG2A and human NKG2C made it a very difficult process to discover anti-NKG2A antibodies that not only bound with high affinity and specificity to the NKG2A protein, but also bound with low or no affinity and specificity to the NKG2C protein.

Surprisingly, the inventors discovered that the 13F3.A4 antibody exhibited certain desired functional characteristics as described in detail herein. FIG. 7A-B show the amino acid sequences of the heavy chain variable region (FIG. 7A) and light chain variable region (FIG. 7B) of the 13F3.A4 anti-NKG2A antibodies. Evidencing the difficulty of discovering anti-NKG2A antibodies that exhibited desired functional characteristics to, for example, treat cancer, even several variants of the 13F3.A4 antibody, which were at first considered potential lead antibodies, ultimately did not exhibit the desired functional properties. Table 2 summarizes certain anti-NKG2A antibodies including variants of the 13F3.A4 antibody that were generated and functionally characterized. Certain variants of the 13F3.A4 antibody, including the NKG2A.9 antibody, unexpectedly exhibited the desired functional characteristics described herein.

TABLE 2 Anti-NKG2A Antibody and Functional Characterization Parental Recombinant Hybridoma Antibody Antibody Description and Functional Clone Name Isotype Characterization 11H9.A4 NKG2A.5 IgG1f Tested in CHO-hNKG2A/HLA- E blocking assay Tested in CHO-cynomolgus NKG2A binding assay. NKG2A.5 showed poor binding to cynomolgus NKG2A expressing CHO cells and was not further developed. FIG. 9A-9B show the heavy chain and light variable region sequences of the 11H9 antibody. 11H9.A4 NKG2A.5 IgG1.3f Tested in CHO-hNKG2A/HLA- E blocking assay Tested in CHO-cynomolgus NKG2A binding assay. Showed poor binding to cynomolgus NKG2A expressing CHO cells and was not further developed. 13F3.A4 NKG2A.6 IgG1.3f Tested in CHO-hNKG2A/HLA- and E blocking assay. NKG2A.6 IgG1f (the parental antibody of, for example, NKG2A.9 and NKG2A. 11) blocked the hNKG2A/HLA-E interaction. Tested in CHO-cynomolgus NKG2A binding assay. NKG2A.6 (the parental antibody of, for example, NKG2A.9 and NKG2A.11) bound to cynomolgus NKG2A Based on the assay results, NKG2A.6-IgG1.3f antibody was further developed and led to the discovery and development of, for example, the NKG2A.9 and NKG2A.11-IgG1.3f antibodies. 13F3.A4 NKG2A.6 IgG2.5 Anti-NKG2A 13F3.A4 with human IgG2-C131S (codon- optimized) Not tested in any assays 13F3.A4 NKG2A.9 IgG1.3f 13F3.A4 with VH-I107T FW VH-I107T, reversion and VK-N30S to VK-N30S remove deamidation site (codon- optimized) Discovered and characterized in numerous functional assays as described in the Examples herein, and surprisingly showed desired functional characteristics. FIG. 15 shows the light and heavy chain amino acid sequences of the NKG2A.9 antibody. 13F3.A4 NKG2A.10 IgG1.3f 13F3.A4 with VH-I107T FW VH-I107T, reversion and VK-N30Q to VK-N30Q remove deamidation site Tested in CHO-hNKG2A/HLA- E blocking and CHO-hNKG2A binding assays 13F3.A4 NKG2A.11 IgG1.3f 13F3.A4 with VH-I107T FW VH-I107T, reversion and VK-N30P to VK-N30P remove deamidation site Tested in CHO-hNKG2A/HLA- E blocking and CHO-hNKG2A binding assays 13F3.A4 NKG2A.12 IgG1.3f 13F3.A4 with VH-I107T FW VH-I107T reversion Tested in CHO-hNKG2A/HLA- E blocking and CHO-hNKG2A binding assays 13F3.A4 NKG2A.13 IgG1.3f 13F3.A4 with N30S to remove VK-N30S deamidation site Not tested in any assays because the NKG2A.13 antibody did not have sufficient yield. 13F3.A4 NKG2A.14 IgG1.3f 13F3.A4 with N30Q to remove VK-N30Q deamidation site Tested in CHO-hNKG2A/HLA- E blocking and CHO-hNKG2A binding assays 13F3.A4 NKG2A.15 IgG1.3f 13F3.A4 with N30P to remove VK-N30P deamidation site Tested in CHO-hNKG2A/HLA- E blocking and CHO-hNKG2A binding assays 2G6.C2 NKG2A.16 IgG1.3f Tested in CHO-cynomolgus and NKG2A binding assay. IgG1f- The antibody showed poor Fab6H binding to cynomolgus NKG2A expressing CHO cells and was not further developed. The antibody also showed increased functionality of NKG2A-negative expressing cells, which suggested that the antibody does not directly affect the NKG2A/HLA-E pathway. FIG. 8A-B shows the heavy chain (FIG. 8A) and light chain (FIG. 8B) amino acid sequences of the 2G6.C2 antibody. 13F3 VH- NKG2A.18 IgG1.3f Hydrophobic patch mutants were I107T, VK- generated to try and reduce N30S-Y49S undesired levels of aggregation. 13F3 VH- NKG2A.19 None of the hydrophobic patch I107T, VK- mutants were tested in any N30S-Y94T assays. 13F3 VH- NKG2A.20 I107T, VK- N30S-Y94A 13F3 VH- NKG2A.21 I107T, VK- N30S-Y94N 13F3 VH- NKG2A.22 Y56T- I107T, VK- N30S 13F3 VH- NKG2A.23 I57T-I107T, VK-N30S 13F3 VH- NKG2A.24 Y58N- I107T, VK- N30S 13F3 VH- NKG2A.25 Y58S- I107T, VK- N30S 27H4.D4 NKG2A.17 mIgG1- 27H4.D4 was re-expressed as D265A NKG2A.17, a chimeric antibody with mouse IgG1-D265A and mouse kappa constant regions. The 27H4.D4 antibody is a non- competing antibody that was generated to bind NKG2A receptor in the presence of the anti-NKG2A antibody of interest, i.e., generated to bind to the same antigen as the target antibody (here, NKG2A), but not to the same epitope. This allowed measurement of the NKG2A receptor expression in the presence of the anti-NKG2A antibody of interest. For the reasons described herein and as shown by the numerous anti-NKG2A antibodies that were not selected for further development due to undesired functional characteristics, generating and discovering anti-NKG2A antibodies with the desired functional properties was an extremely difficult process. For example, even during advanced stages of anti-NKG2A antibody development, the 2G6.C2, 4G5.D1, 11H9.A4 and 1G5.B2 antibodies, which were initially considered potential lead antibodies, were ultimately not selected for further development because they did not exhibit all of the desired functional properties. FIG. 8A-B show the sequences of the heavy and light chain variable region of the 2G6.C2 antibody. FIG. 9A-B show the sequences of the heavy and light chain variable region of the 11H9.A4 antibody. FIG. 10A-B show the sequences of the heavy and light chain variable region of the 4G5.D1 antibody. FIG. 11A-B show the sequences of the heavy and light chain variable region of the 1G5.B2 antibody. These examples, amongst others described herein, demonstrate the difficulty of generating anti-NKG2A antibodies with the desired functional characteristics, including specific binding to NKG2A protein and not to NKG2C protein. As explained herein, part of the difficulty in discovering anti-NKG2A antibodies with desired functional properties was due to the high sequence homology between the human NKG2A and human NKG2C proteins.

Variants of the 13F3.A4 antibody were discovered and further characterized. As shown in FIGS. 12-13, sequence liabilities were assessed in the 13F3.A4 antibody. The variable sequence of the fully human anti-NKG2A monoclonal antibody NKG2A.9 was derived from the 13F3.A4 hybridoma with a VH-I107T germline reversion mutation and a VK-N30S mutation to mitigate the deamidation liability of the VK-N30 residue. The constant region was derived from an IgG1f backbone (IgG1.3) and includes three mutations on the heavy chain: L234A, L235E, and G237A to minimize or eliminate antibody binding to Fcγ receptors and C1q. The M34 oxidation was identified as a potential risk for oxidation but was de-risked based on subsequent accelerated stability studies. The full length amino acid sequence of the NKG2A.9 antibody is shown in FIG. 14. The NKG2A.9 antibody was selected and tested in numerous functional assays as described herein to show safety and efficacy alone and in combination with other agents to treat, for example, cancer.

The full length amino acid sequence of the NKG2A.11 antibody is shown in FIG. 15. NKG2A.11 is derived from 13F3.A4 with a VH-I107T framework reversion and VK-N3OP to remove the deamidation site. There is one amino acid difference between NKG2A.9 (N30S) and NKG2A.11 (N30P) in the light chain, and no amino acid differences in the heavy chain. NKG2A.9 and NKG2A.11 have similar thermal stability and solubility properties. NKG2A.9 has better thermal reversibility and better immunogenicity (in silico) than NKG2A.11. The immunogenicity of NKG2A.9 is described in more detail in Examples 16 and 26.

(2) Hundreds of Antibodies Generated Using Single B Cell Cloning Method Lacked Desired Functional Characteristics

FIG. 16 shows a circle plot epitope binning results of exemplary antibodies generated by the B cell cloning method. The epitope binning was performed on an Octet HTX instrument using the “in-tandem format.” First, hNKG2A-hCD94-mFc fusion protein was captured on Octet sensor tips. Next, the remaining capture sites were blocked if required. Third, the antigen was saturated with single B cell cloning-derived antibodies from supernatant. Finally, the binding of reference antibodies was tested. The epitope binning experiment was repeated with reverse order of addition, saturating first with reference antibodies, and then probing for binding of supernatant containing single B cell cloning-derived antibodies. This epitope binning showed high diversity in binding and blocking behavior, corresponding to high diversity in the epitopes. FIG. 16 depicts the epitope binning results by displaying antibodies that cross-block each other connected by a line. Sample antibodies that have a similar blocking profile as compared to the benchmark antibodies (13F3.A4, Z270, RD-ahNKG2a (clone 131411, Catalog No. MAB1059), and RD-ahCD94 (clone 131412, Catalog No. MAB1058)) are grouped together into groups 1-4, 6-7, and 9. Benchmark antibodies with similar blocking profiles as the sample antibody sets are also grouped together into groups 5, 8 and 10. The antibodies in the same group could potentially cross-block each other as well, but this was not tested these experiments. Sample antibodies were not tested against each other, and benchmark antibodies were not tested against each other for cross-blocking.

The ability of anti-human NKG2A antibodies generated using the single B cell cloning method to block NKG2A/HLA-E interaction was assessed. As depicted in FIG. 17A, 1×10⁶ human NKG2A-expressing CHO cells were incubated with 100 μL supernatant or 10 μg/mL purified antibody for 30 minutes at 4° C. Cells were washed and stained with fluorescently labeled HLA-E pentamer (Proimmune) to detect bound HLA-E on the cell surface. Cells were read fresh on the BD LSRFortessa. FIG. 17B shows the ability of anti-human NKG2A antibodies generated by B cell cloning to block HLA-E binding to human NKG2A-expressing CHO cells. The NKG2A.9 antibody served as the positive control, and the isotype served as the negative control. The hundreds of antibodies generated using the SBCC method only partially blocked or did not block the NKG2A/HLA-E interaction. None of these hundreds of antibodies were selected for further development, providing evidence of the difficulty of discovering anti-NKG2A antibodies with the desired functional characteristics.

(3) Anti-Human NKG2A Antibodies that Desirably Blocked the NKG2A/HLA-E Interaction and not the NKG2C/HLAE-Interaction

The ability of anti-human NKG2A antibodies to block the NKG2A/HLA-E and NKG2C/HLA-E interactions was assessed. As shown in FIG. 18D, 1×10⁶ human NKG2A-expressing CHO cells were incubated with titrating amounts of anti-NKG2A antibody for 30 minutes at 4° C. Cells were washed and stained with fluorescently labeled HLA-E pentamer (Proimmune) to detect bound HLA-E on the cell surface. Cells were read fresh on the BD LSRFortessa. FIG. 18E-F show the HLA-E blocking curve and IC₅₀ of anti-NKG2A antibodies in blocking HLA-E binding to human NKG2A-expressing or NKG2C-expressing CHO cells. The 13F3.A4 and 11H9.A1 antibodies desirably showed specific binding to human NKG2A-expressing CHO cells (FIG. 18E), and desirably did not block the NKG2C/HLA-E interaction (FIG. 18F). However, the 2EB.B1 antibody undesirably bound to human NKG2C-expressing CHO cells and blocked the NKG2C/HLA-E interaction. Thus, the 2EB.B1 antibody was not selected for further development.

The ability of anti-NKG2A antibodies to block the NKG2A/HLA-E interaction in natural killer cells (NKLs) was also assessed. As shown in FIG. 19C, 1×10⁶ endogenously expressing human NKL cells were incubated with titrating amounts of anti-NKG2A antibody for 30 minutes at 4° C. Cells were washed and stained with fluorescently labeled HLA-E pentamer (Proimmune) to detect bound HLA-E on the cell surface. Cells were read fresh on the BD LSRFortessa. FIG. 19D shows the HLA-E blocking curve and IC₅₀ values of anti-NKG2A antibodies in blocking HLA-E binding to human NKG2A expressing NKL cells. The 13F3.A4, 11H9.A1, 4G5.D1, and IG5.B2 antibodies showed blocking of the NKG2A/HLA-E interaction in NKL cells with IC₅₀ values of 0.2 nM, 0.1 nM, 0.3 nM, and 0.4 nM, respectively.

The ability of additional anti-human NKG2A antibodies to block the NKG2A/HLA-E interaction was assessed. As depicted in FIG. 20A, 1×10⁶ human NKG2A-expressing CHO cells were incubated with titrating amounts of anti-NKG2A antibody (10 μg/ml) for 30 minutes at 4° C. Cells were washed and stained with fluorescently labeled HLA-E pentamer (Proimmune) to detect bound HLA-E on the cell surface. Cells were read fresh on the BD LSRFortessa. FIG. 20B-C show the HLA-E blocking curve, and IC₅₀ values of anti-human NKG2A antibodies in their ability to block HLA-E binding to human NKG2A-expressing CHO cells. As shown in FIG. 20B, the NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, and NKG2A.15 antibodies all demonstrated relatively low IC₅₀ values (0.1, nM, 0.05 nM, 0.1 nM, 0.1 nM, 0.2 nM, and 0.1 nM, respectively), which indicated that the antibodies blocked the NKG2A/HLA-E interaction. Similarly, as shown in FIG. 20C, the NKG2A.5, 4G5.D1, and 1G5.B2 antibodies demonstrated relatively low IC₅₀ values of 0.3 nM, 0.9 nM, and 0.7 nM, respectively. Thus, these antibodies were selected for further development. However, as described herein, the NKG2A.5 and 4G5.D1 antibodies were later eliminated for further development due to their poor binding to cynomolgus NKG2A protein.

As shown in FIG. 20C, however, the 25E7.G8 antibody (depicted with a closed circle) did not block the NKG2A/HLA-E interaction, as shown by the relatively high IC₅₀ value of about 4 nM. Thus, the 25E7.G8 antibody was not selected for further development.

(4) Anti-NKG2A Antibodies that Showed Desirable Specific Binding to Human NKG2A-Expressing CHO Cells.

As depicted in FIG. 18A, 1×10⁶ human NKG2A-expressing or human NKG2C-expressing CHO cells were incubated with titrating amounts of anti-NKG2A antibody for 30 minutes at 4° C. Cells were washed and stained with fluorescently labeled goat anti-human IgG-PE secondary antibody for 30 minutes at 4° C. to detect the bound antibody on the cell surface. Cells were read fresh on the BD LSRFortessa. FIG. 18B-C show the binding curve and EC₅₀ (nM) of anti-NKG2A antibodies to human NKG2A-expressing or NKG2C-expressing CHO cells. The 13F3.A4, 11H9.A1, and 2EB.B1 antibodies showed specific binding to the human NKG2A-expressing CHO cells, a shown by the EC₅₀ values of 0.7 nM, 0.7 nM, and 0.3 nM, respectively.

As shown in FIG. 21A, 1×10⁶ human and cynomolgus NKG2A expressing CHO cells were incubated with titrating amounts of antibody (10 μg/ml) for 30 minutes at 4° C. Cells were washed and stained with fluorescently labeled goat anti-human IgG-PE secondary antibody for 30 minutes at 4° C. to detect the bound antibody on the cell surface. Cells were read fresh on the BD LSRFortessa. FIG. 21B-C show the binding curves, and EC₅₀ values of anti-human NKG2A antibodies to human NKG2A-expressing CHO cells.

As shown in FIG. 21B, the NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, and NKG2A.15 antibodies all demonstrated relatively low EC₅₀ values (0.1 nM, 0.1 nM, 0.1 nM, 0.2 nM, 0.1 nM, and 0.1 nM, respectively), which indicated that the anti-NKG2A antibodies desirably bound with specificity to the NKG2A receptor in human NKG2A-expressing CHO cells. Similarly, as shown in FIG. 21C, the NKG2A.5, 4G5.D1, and 1G5.B2 antibodies demonstrated relatively low EC₅₀ values (0.3 nM, 0.6 nM, and 0.6 nM, respectively), which indicated the antibodies bound to the NKG2A receptor. Thus, the NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, NKG2A.15, NKG2A.5, 4G5.D1, and 1G5.B2 were selected for further development at this stage.

(5) Anti-NKG2A Antibodies that Bound to Cynomolgus NKG2A-Expressing CHO Cells

The ability of anti-NKG2A antibodies that bound to cynomolgus NKG2A-expressing CHO cells was assessed. As depicted in FIG. 22A, 1×10⁶ cynomolgus NKG2A-expressing CHO cells were incubated with titrating amounts of anti-NKG2A antibody (10 μg/ml) 10 for 30 minutes at 4° C. Cells were washed and stained with fluorescently labeled HLA-E pentamer (Proimmune) to detect bound HLA-E on the cell surface. Cells were read fresh on the BD LSRFortessa. FIG. 22B-C show the binding curves, and EC₅₀ values of anti-human NKG2A antibodies to cynomolgus NKG2A-expressing CHO cells.

As shown in FIG. 22B, the NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, and NKG2A.15 antibodies all demonstrated relatively low EC₅₀ values (1.2 nM, 1.3 nM, 1.4 nM, 1.2 nM, 1.2 nM, and 1.5\nM, respectively), which indicated that the anti-NKG2A antibodies bound to the NKG2A receptor in cynomolgus monkey NKG2A-expressing CHO cells.

As shown in FIG. 22C, the NKG2A.5, 4G5.D1, and 1G5.B2 antibodies showed poor binding to cynomolgus monkey NKG2A expressing CHO cells, as demonstrated by the relatively high EC₅₀ values of 172 nM, about 314 nM, and 21 nM, respectively. Thus, these three antibodies were not selected for further development. The 2G6.C2 antibody had an EC₅₀ value of 0.6 nM, which desirably showed specific binding to cynomolgus monkey NKG2A expressing CHO cells; thus, the 2G6.C2 antibody was selected for further development.

The ability of the 2G6.C2 antibody to specifically bind to the NKG2A receptor and to block the NKG2A/HLA-E interaction was assessed using the same methods as described above in this Example 3(3) and 3(4), and as depicted in FIGS. 23A and 23C. The 2G6.C2 antibody was comparable to, for example, the NKG2A.9 antibody in its ability to block the NKG2A/HLA-E interaction, as shown by the similar IC₅₀ values of 1.1 nM for the NKG2A.9 antibody and 1.3 nM for the 2G6.C2 antibody, as shown in FIG. 23B. Similarly, the 2G6.C2 antibody was comparable to, for example, the NKG2A.9 antibody in its ability to bind to the NKG2A receptor in human NKG2A-expressing CHO cells, as shown by the similar EC₅₀ values of 0.8 nM for the NKG2A.9 antibody and 1 nM for the 2G6.C2 antibody, as shown in FIG. 23D.

Despite being able to desirably block the NKG2A/HLA-E interaction and to bind to the NKG2A receptor, the 2G6.C2 antibody was not selected for further development because the 2G6.C2 non-specifically enhanced functionality among NKG2A negative (−) NK cells. This demonstrates the difficulty of discovering anti-NKG2A antibodies with the desired functional characteristics described herein to treat cancer.

(6) Anti-NKG2A Antibodies that Bound to Human NKG2A+ Natural Killer Cell Line (NKL)

As shown in FIG. 19A, 1×10⁶ endogenously expressing NKG2A human NKL cells were incubated with titrating amounts of anti-NKG2A antibody for 30 minutes at 4° C. Cells were washed and stained with fluorescently labeled goat anti-human IgG-PE secondary antibody for 30 minutes at 4° C. to detect the bound antibody on the cell surface. Cells were read fresh on the BD LSRFortessa. FIG. 19B shows the binding curve and EC₅₀ values (nM) of anti-NKG2A antibodies to human NKG2A expressing NKL cells. The EC₅₀ values for the 13F3.A4, 11H9.A1, 4G5.D1, and IG5.B2 antibodies were 0.2 nM, 0.1 nM, 0.2 nM, and 0.3 nM, respectively, and these values indicated that these anti-NKG2A antibodies desirably bound specifically to the NKG2A+ natural killer cells.

(7) Anti-NKG2A Antibodies that Bound to Cynomolgus NKG2A+ NKL

As depicted in FIG. 24A, 1×10⁶ peripheral blood mononuclear cells (PBMC) isolated from cynomolgus monkeys were incubated with titrating amounts of anti-NKG2A antibody for 30 minutes at 4° C. Cells were washed and stained with fluorescently labeled goat anti-human IgG-PE secondary antibody for 30 minutes at 4° C. to detect the bound antibody on the cell surface. Cells were read fresh on the BD LSRFortessa. FIG. 24B shows the binding curve and EC₅₀ values (nM) of anti-NKG2A antibodies to cynomolgus NKG2A expressing NK cells. As shown in FIG. 24B, the 11H9.A1 and 4G5.D1 antibodies did not bind to cynomolgus NKG2A+NK cells, as indicated by EC₅₀ values of 28 nM and 38 nM, respectively. Thus, because of their lack of binding to cynomolgus monkey NKG2A+NK cells, the 11H9.A1 and 4G5.D1 antibodies were eliminated from further development to treat, for example, cancer. The 13F3.A4 antibody desirably bound to cynomolgus monkey NKG2A+NK cells, as indicated by the EC₅₀ value of 0.2 nM. Thus, the 13F3.A4 antibody demonstrated desirable binding functionality and was selected for further modification and development.

(8) Anti-NKG2A Antibodies that Enhanced Natural Killer (NK) Cell Response of Degranulation

In in vitro experiments using peripheral blood mononuclear cells (PBMC) from healthy human donors, anti-NKG2A antibody blockade of NKG2A/HLA-E interaction among activated NK cells increased NK cell degranulation. One of the mechanisms by which NK cells eliminate their targets, such as tumor cells, is through a complicated, multi-stage process that concludes in the directed secretion of granules containing the lytic enzymes perforin and granzymes. This cellular process of directed secretion of molecules from granules is known as degranulation. After NK cells release secretory lytic granules to, for example, the target tumor cells, perforin generates pores in the target tumor cell membranes, and granzymes then access the target tumor cell cytoplasm and induce tumor cell death (also known as apoptosis). During this process, the lysosome-associated membrane protein-1 (LAMP-1, also known as CD107A) is transported to the surface of the NK cell, which renders it accessible for antibody binding, thus making it possible to identify NK cells that have been active in degranulation. We then measured the effect of the anti-NKG2A antibodies disclosed herein on NK cell activation and enhancement of NK cell functionality in the degranulation process, which results in increased tumor cell death. This process is partially illustrated in FIG. 25A. The X in the NK cell illustrates the inhibitory signal when NKG2A interacts with HLA-E. The dark circles in the NK cell represent granules.

NK cells isolated from normal PBMC donors were activated overnight with recombinant human (rh) interleukin-2 (IL-2). Specifically, NK cells were isolated from human whole blood using Ficoll gradient and the human NK cell isolation kit (Miltenyi Biotech). NK cells were cultured overnight with rh IL-2 (400 IU/ml). Following activation, the cell viability was more than 90%, as determined by cell counter. NK cells were then co-cultured for four hours with 5×10⁴ of the human B-lymphoblastoid target cell line 721.221 expressing HLA-E in the presence of anti-human NKG2A antibodies (the NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.14, NKG2A.15, NKG2A.5, 2G6.C2, 4G5.D1, 25E7.G8) and an isotype antibody (human IgG1.3). Cells were then collected and stained for the following markers: anti-human CD3, anti-human CD56, live/dead, and anti-human CD107a, and non-competing anti-human NKG2A antibody (the 27H4.D4 clone). Cells were acquired fresh on the BD LSRFortessa™. Percent degranulation (% CD107) among NKG2A+ or NKG2A− NK cells was subsequently analyzed on FlowJo Version 10 (flow cytometry software). Fold increase was calculated by increase in % CD107a over the isotype control.

In the presence of anti-human NKG2A monoclonal antibodies, there was an increase in NK cell degranulation as measured by CD107a expression by flow cytometry. The increase in NK cell response was not observed on NKG2A negative (−) NK cells, which showed that the antibody effect was specific to NKG2A expressing NK cells.

FIGS. 25B-C are graphical representations of the flow cytometry analyses, and shows that all tested anti-NKG2A antibodies (the NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, NKG2A.15, NKG2A.5, 2G6.C2, and 4G5.D1 antibodies) enhanced NK cell degranulation (as measured by fold change in % CD107a compared to the isotype control) among NKG2A+ NK cells, except the 25E7.G8 clone (a non-blocking antibody) (as shown in FIG. 25B), which was included as a negative control. (In other words, it was expected that the 25E7.G8 antibody would not increase NK cell degranulation because the antibody does not block the NKG2A/HLA-E pathway).

Specifically, in assessing NK cells from several donors with a fixed concentration of 10 μg/ml of the NKG2A.9 antibody, there was an approximately two-fold increase in percent degranulation among NKG2A+ expressing NK cells compared to the human IgG1.3 isotype control. As shown in FIG. 25C, for the NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, NKG2A.15, NKG2A.5, and 4G5.D1 antibodies, the increase in NK cell response was not observed on NKG2A negative (−)(also referred to herein as NKG2A−) NK cells, which showed that the % CD107 increase was specific to NKG2A expressing NK cells. In other words, for several of the anti-NKG2A antibodies (the NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, NKG2A.15, NKG2A.5, 4G5.D1 antibodies), enhanced degranulation among NKG2A positive(+) (also referred to herein as NKG2A+) NK cells and lack of enhanced degranulation among NKG2A negative (−) NK cells showed that NKG2A receptor expression was needed to enhance functionality. One exception to this result, as shown in FIG. 25C, was the 2G6.C2 antibody, which showed enhanced NK cell degranulation even among NKG2A negative (−) NK cells. In other words, enhanced degranulation with the 2G6.C2 antibody was not specific to the NKG2A/HLA-E interaction. Thus, the 2G6.C2 antibody was not selected for further investigation.

In summary, the NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, NKG2A.15, NKG2A.5, and 4G5.D1 antibodies surprisingly and desirably specifically enhanced degranulation among NKG2A-expressing NK cells as compared to the isotype control. As shown in FIG. 25B, for the NKG2A.9 antibody, this enhanced degranulation was approximately two-fold compared to the isotype control. However, other anti-NKG2A antibodies were not selected for further development because they did not exhibit desired functional characteristics. For example, the 2G6.C2 antibody resulted in increased functionality of NKG2A negative (−) expressing cells, which suggested that the 2G6.C2 antibody did not directly affect the NKG2A/HLA-E interaction. The P1-069366 antibody also did not show functionality in the NK degranulation assay. As shown in FIG. 53, the P10069366 antibody had a similar % CD107⁺ within NKG2A⁺ NK cells are the isotype. Thus, the 2G6.C2 and P1-069366 antibodies were not selected for further development because they did not enhance degranulation in NK cells.

(9) Anti-NKG2A Antibodies that Enhanced NK Cell Response of Increased Interferon-Gamma (IFNγ) Production.

Anti-human NKG2A antibodies increased IFN-γ production in NKL cells co-cultured with CHO/MICA/HLA-E (CHO cells engineered to express HLA-E and MICA, the ligand for NK cell activating receptor NKG2D). As illustrated in FIG. 26A, in in vitro experiments using NKL cells and CHO/MICA/HLA-E, anti-NKG2A antibody blockade of the NKG2A/HLA-E interaction among activated NK cells (via the MICA-NKG2D pathway) desirably increased IFN-γ production. We first activated NKL cells using transfected MICA on CHO cells so that the activated NKL cells can kill the target cells, CHO/MICA/HLA-E cells. MICA functions as a ligand recognized by the activating receptor NKG2D that is expressed on the surface of NK cells. The antibodies were evaluated in an assay using a co-culture of NKL and CHO/MICA/HLA-E cells. In this assay, MICA on CHO cells served to activate NKL cells via NKG2A (positive signal), and the expression of HLA-E on CHO cells inhibits NKG2A expressing NKL cells (negative signal). The assay tested whether the anti-NKG2A antibody would block the NKG2A/HLA-E interaction and desirably increase IFN-γ production.

Specifically, NKL cells were co-cultured with CHO/MICA/HLA-E at an effector cell-to target cell ratio (E:T) of 4:1 in the presence of 10 μg/mL anti-human NKG2A antibody or isotype control antibody. Following overnight stimulation in a 37° C. incubator, IFN-γ production was measured in the supernatant by ELISA. As shown in FIG. 26B, the 13F3.A4, 11H9.A1, and 4G5.D1 antibodies desirably increased IFN-γ production as compared to the isotype control (human IgG1.3). The 25E7.G8 antibody, an anti-human NKG2A antibody that does not block NKG2A/HLA-E interaction, was used as a negative control, and, as expected, did not increase IFN-γ production compared to the isotype control.

(10) Anti-NKG2A Antibodies that Enhanced CD8+ T Cell Response of Increased IFNγ Production.

Anti-human NKG2A antibodies increased IFN-γ production among CD8+ T cells co-cultured with the pancreatic carcinoma cell line, Hs766T.

Peripheral blood mononuclear cells (PBMC) from normal donors were activated for three days with plate-bound CD3 (OKT3) and rhIL-15 to increase expression of NKG2A among CD8+ T cells. As depicted in FIG. 27A, CD8+ T cells were co-cultured with Hs766T target cells (treated with mitomycin C) at an effector cell-to target cell ratio (E:T) of 1:1 in the presence of 10 μg/mL anti-human NKG2A antibodies or isotype control antibody (human IgG1.3). After five days of stimulation at 37° C., IFN-γ production was measured in the supernatant by ELISA.

As shown in FIG. 27B, the 13F3.A4 and 11H9.A1 antibodies increased IFN-γ production compared to the isotype control. The 25E7.G8 antibody, an anti-human NKG2A antibody that does not block NKG2A/HLA-E interaction, was used as a negative control and, as expected, did not increase IFN-γ production over isotype control. Specifically, HS766T is a pancreatic carcinoma cell line that endogenously expresses HLA-E. CD8+ T cells from healthy donor PBMC generally express low levels of NKG2A. However, with anti-CD3 and IL-15 stimulation, we were able to increase NKG2A expression on these cells. In this system, the CD8+ T cells express NKG2A, and the target HS766T cells express HLA-E. The 13F3.A4 and 11H9.A1 antibodies blocked NKG2A/HLA-E interaction and increased the cytotoxic functionality of CD8+ T cells, which was demonstrated by an increase in IFN-γ production.

(11) 13F3.A4 Antibody and Variants, Including the NKG2A.9 Antibody, Demonstrated Safe Levels of Undesirable Antibody Aggregation

Antibody aggregates are clusters of denatured antibody molecules that are irreversibly formed during antibody expression in the cell culture, product purification, or storage as the antibody drug product. The process of aggregation is complicated and influenced by the biochemical and biophysical properties of the antibody as well as the physicochemical environmental in which the antibody is exposed during processing and storage. Antibody aggregation may expose normally unexposed epitopes, leading to increase immunogenicity. Purification processes for antibody products aims to achieve low aggregate levels, for example, less than 2% soluble aggregates. Accelerated stability studies (also known as forced degradation studies) were performed on eight variants of the 13F3.A4 antibody (NKG2A.9, NKG2A.13, NKG2A.18, NKG2A.19, NKG2A.21, NKG2A.22, NKG2A.23, and NKG2A.24) to ascertain the aggregation propensity of these antibodies at 4° C. and 25° C. storage for one month. NKG2A.20 stability was tested only at 4° C. due to limited material availability. A formulation with 20 mM histidine (pH 6.0), 260 mM sucrose, 50 μM DTPA and 0.05% polysorbate 80 was used as the test formulation for all the antibodies. Soluble aggregates were monitored by size exclusion chromatography (SEC) analysis. The tested concentrations were based on material availability. SEC revealed comparable aggregation propensities for all the antibodies except NKG2A.19, where multiple peaks are observed. Although not bound by any other mechanism, NKG2A.19 may have undergone degradation due to the presence of significant levels of protease in the batch of material tested.

Overall, the aggregation levels (less than 2% soluble aggregates at 25° C. after one-month storage) indicated suitability to formulate at pH 6.0 for the other antibodies that were tested. The physical stability data demonstrated less than 2% soluble aggregates after one month of storage at 25° C., which indicated that the tested formulation with the anti-NKG2A antibodies were suitable as a preliminary formulation imparting acceptable storage stability for drug substance at the tested concentration.

Tested Soluble aggregate (%) NKG2A concentration 4° C. for 25° C. for sequence (mg/mL) one month one month NKG2A.9 150 0.7 0.8 NKG2A.18 90 Not 1.2 detected NKG2A.19 54 Degraded sample NKG2A.20 6 1.2 Not tested NKG2A.21 43 1.3 1.9 NKG2A.22 120 0.8 1.2 NKG2A.23 130 0.8 1.1 NKG2A.24 120 0.8 1.1 (12) NKG2A.9 Antibody Bound with High Affinity to NKG2A Versus NKG2C Protein

The specificity of NKG2A.9 antibody for NKG2A protein over NKG2C protein was assessed by determining the kinetics and affinities using a Biacore T200 SPR instrument. The extracellular domains of NKG2A protein and NKG2C protein were each prepared as a heterodimer with the extracellular domain of CD94. The assay temperature was 37° C., and the running buffer was HEPES buffered saline (10 mM HEPES, 150 mM NaCl) at pH 7.4 supplemented with 0.05% Tween-20 and 1 g/L Bovine Serum Albumin (BSA). The NKG2A.9 antibody was captured on a CM4 sensor chip with pre-immobilized anti-human Fc capture reagent (Southern Biotech Catalog Number 2081-01). The NKG2A-CD94 and NKG2C-CD94 heterodimers were flowed as analytes over the captured antibody in two five-membered, three-fold dilution series each. One concentration series was used for single-cycle kinetics and the other one for multi-cycle kinetics. For the multi-cycle kinetics analysis, the second highest concentration was injected in duplicate. The top concentration for NKG2A-CD94 was 250 nM, and the top concentration for NKG2C-CD94 was 1.5 uM. All data were double-referenced and fitted to a 1:1 binding model with mass transport using the Biacore T200 Evaluation Software version 3.1

Generally, as discussed herein, two kinetic parameters determine binding affinity. The first parameter is how fast the complex is formed (the association rate, k_(a)), and the second parameter is how fast the complex dissociates or falls apart (the dissociation rate, k_(d)). Both kinetic parameters can be summarized in the equilibrium dissociation constant (K_(D)), which is defined as k_(d)/k_(a). This comparative Biacore experiment addressed both interaction parameters: how fast the NKG2A.9 antibody/NKG2 protein complex was formed (k_(a)) and how fast it fell apart (k_(d)).

In this comparative Biacore experiment, the NKG2A.9 antibody showed 15-fold stronger binding to NKG2A protein than to NKG2C protein (K_(D) of 3.9×10⁻⁸M for binding to NKG2A versus K_(D) of 5.7×10⁻⁷ M for binding to NKG2C). In other words, the NKG2A.9 antibody showed 15-fold weaker binding to NKG2C protein than to NKG2A protein. These results indicated that NKG2A.9 antibody had high selectivity for NKG2A protein compared to NKG2C protein, as summarized in the Table below. The NKG2A.9 antibody's stronger binding to NKG2A protein was driven by a faster association rate (ka), that is, how fast the NKG2A.9 antibody/NKG2A protein complex formed compared to the NKG2A.9 antibody/NKG2C protein complex. In other words, the NKG2A.9 antibody formed a complex 15 times more quickly with NKG2A protein than with NKG2C protein.

TABLE Comparative Affinities of NKG2A.9 Antibody for NKG2A and NKG2C Proteins K_(D) ratio Ligand Sample k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) (NKG2C/NKG2A) NKG2A.9 hNKG2A- 3.9 × 10⁵ 1.5 × 10⁻² 3.9 × 10⁻⁸ heterodimer NKG2A.9 hNKG2C- 2.4 × 10⁴ 1.4 × 10⁻² 5.7 × 10⁻⁷ 15 heterodimer

FIG. 56A-D show the binding affinity of the NKG2A.9 antibody to human NKG2A-CD94 heterodimers (FIG. 56A-B) and NKG2C-CD94 heterodimers (FIG. 56C-D) at 37° C. as determined by Biacore using both single-cycle kinetics (FIGS. 56A and 56C) and multi-cycle kinetics (FIGS. 56B and 56D). The SPR response is shown for analyte association and dissociation.

In other embodiments, the NKG2A.9 antibody forms a complex with NKG2A protein 14 times more quickly, 13 times more quickly, 12 times more quickly, 11 times more quickly, 10 times more quickly, 9 times more quickly, 8 times more quickly, 7 times more quickly, 6 times more quickly, 5 times more quickly, 4 more times more quickly, three time more quickly, or two times more quickly with than with NKG2C protein.

(13) Summary of Surprising and Desirable Functional Properties of Anti-NKG2A Antibodies

In summary, the inventors unexpectedly discovered certain anti-NKG2A antibodies that exhibited various desired functional properties for treatment of, for example, cancer. In certain embodiments, these anti-NKG2A antibodies include the 13F3.A4 antibody clone and variants, including the NKG2A.9 and NKG2A.11 antibodies, which were identified and selected from hundreds of antibodies that did not exhibit the desired functional properties. Specifically, the NKG2A.9 antibody is an anti-hNKG2A antibody that is also known as 13F3.A4-VH-I107T-N3OS IgG1.3, and will be referred to herein as the “NKG2A.9” antibody. Mutations in residue VH-107T (framework mutation reverted to germline) and residue VK-N3OS to lower deamidation potential and reduce immunogenicity risk resulted in the discovery of the NKG2A.9 antibody. As described herein and shown in FIG. 13, various sequence liabilities were assessed in the 13F3.A4 antibody that led to the optimization and development of, for example, the NKG2A.9 antibody.

As described in further detail in Examples herein, anti-NKG2A antibodies, including the NKG2A.9 antibody, demonstrated, for example, the following surprising and desirable functional characteristics:

-   -   a) Blocked the NKG2A/HLA-E interaction;     -   b) Reversed NKG2A-mediated inhibitory signaling;     -   c) Did not bind, or bound with low affinity to, cells expressing         human NKG2C protein;     -   d) Bound with high affinity to cells expressing human and         cynomolgus NKG2A; and/or     -   e) Bound to human and cynomolgus NKG2A+ natural killer cells     -   f) Enhanced natural killer cell response, for example, of         increased interferon-gamma (IFNγ); and/or     -   g) Enhanced CD8+ T cell response, for example, of increased IFNγ         production; and/or     -   h) Formed a complex more quickly with NKG2A protein than with         NKG2C protein; in some embodiments, formed a complex 15 times         more quickly with NKG2A protein than with NKG2C protein.

The NKG2A.9 antibody was characterized and tested alone and in combination with additional antibodies as described in the Examples herein. The NKG2A.9 antibody bound with high affinity and specificity to human NKG2A protein. The EC₅₀ values for the binding of NKG2A.9 to NKG2A protein were as follows and demonstrated binding of NKG2A.9 to NKG2A protein:

Cell lines EC₅₀ NKL (human NKG2A endogenously expressed 0.4 nM on an NK cell line ) CHO-human NKG2A (human NKG2A ectopically 0.6 nM expressed on CHO cells) CHO-cyno NKG2A (cynomolgus monkey NKG2A 1.2 nM ectopically expressed on CHO cells) The EC₅₀ values for the binding of NKG2A.9 to NKG2C protein were as follows and desirably demonstrated lack of binding of NKG2A.9 to human NKG2C protein:

Cell lines EC₅₀ CHO-human NKG2C (human NKG2C ectopically 9.0 nM expressed on CHO cells) CHO-cyno NKG2C (cynomolgus monkey NKG2C 0.8 nM ectopically expressed on CHO cells) The IC₅₀ values of NKG2A.9, which showed blocking of the NKG2A/HLA-E interaction in cell blocking assays, were as follows:

Cell lines IC₅₀ NKL 0.3 nM CHO-human NKG2A 1.0 nM The EC₅₀ values showed that the NKG2A.9 antibody blocked the NKG2A/HLA-E interaction. The EC₅₀ value of the NKG2A.9 antibody showed that binding to human NKG2A protein was about 15-fold less than to human NKG2C protein (0.6 nM for NKG2A vs. 9.0 nM for NKG2C). Furthermore, no specific binding of the NKG2A.9 antibody to human NKG2C was observed in SPR experiments. In contrast, the EC₅₀ values the NKG2A.9 antibody binding to cynomolgus NKG2A and cynomolgus NKG2C proteins were similar (1.2 nM for NKG2A vs. 0.8 nM for NKG2C). The NKG2A.9 antibody did not block the human NKG2C/HLA-E interaction as assessed by flow cytometry under the conditions discussed herein. No binding to mouse or rat NKG2A was observed by flow cytometry on primary NK cells.

We used Scatchard analysis to measure binding affinities of NKG2A.9. For NKG2A.9, saturated cell binding and Scatchard analysis of CHO-hNKG2A and CHO-cynomolgus NKG2A established specific binding affinities (K_(D) values) of about 0.4 nM and 1.0 nM, respectively, as shown in FIG. 52A-B. Specifically, NKG2A.9 was radioiodinated with ¹²⁵I—Na (1 mCi; PerkinElmer Catalog NEZ033H001 MC) using IODO-GEN® solid phase iodination reagent (1,3,4,6-tetrachloro-3a-6a-diphenylglycouril; Pierce, Catalog 28601). Excess iodide was removed using a desalting column (Pierce, Catalog 43243). Fractions of labeled antibody were collected and analyzed for radioactivity on a Wizard 1470 gamma counter. The ¹²⁵I-NKG2A.9 concentration in each fraction was calculated with the Qubit fluorometer from Invitrogen. Radiopurity was established by thin layer chromatography of peak protein and radioactive fractions. Radioiodinated NKG2A.9 binding to CHO over-expressed human or cynomolgnus NKG2A cells was demonstrated by incubating CHO-human or cynomolgus NKG2A cells with a titration of 125I-NKG2A.9. Nonspecific binding was determined by binding in the presence of a titration of a 100-fold molar excess of unlabeled antibody and was subtracted from total CPM to calculate specific binding. A linear standard curve of 125I-NKG2A.9 concentration versus CPM was used to extrapolate specific activity, maximal nM bound 125I-NKG2A.9 and thereby calculate receptor number per cell. Results of the Scatchard analysis showed that NKG2A.9 specifically bound to CHO-hNKG2A with an equilibrium dissociation constant (K_(D)) of 0.4 nM and to CHO-cynoNKG2A transfectants with a K_(D) of 0.1 nM. The relative binding affinity of NKG2A.9 for cynomolgus NKG2A was determined via SPR by flowing NKG2A.9 Fab over a CM4 chip with immobilized monkey NKG2A-CD94-mFc (truncated construct to achieve expression). Human NKG2A-CD94-mFc coupled to a second flow cell served as a control. Another flow cell was left blank for reference-subtraction. The measurement was conducted on a Biacore T200 instrument at 37° C. using Hepes buffered saline (10 mM HEPES, 150 mM NaCl) at pH 7.4 supplemented with 0.05% Tween-20 and 1 g/L BSA as running buffer. A concentration series of NKG2A.9 Fab was injected over the different flow cells. All data were double-referenced and fitted to a 1:1 Langmuir model using the Biacore T200 Evaluation Software version 3.1. In this assay, the monovalent binding of NKG2A.9 Fab to human NKG2A was approximately 26-fold stronger than binding to cynomolgus NKG2A. The K_(D) value as measured by SPR for human NKG2A was 61 nM, and K_(D) value as measured by SPR for cynomolgus NKG2A was 1600 nM, as shown in the table below. These K_(D) values indicated that the NKG2A.9 Fab binds to recombinant cynomolgus NKG2A, but higher Fab concentrations have to be used to achieve a binding level (saturation level) similar to that of human NKG2A.

hNKG2A-CD94 cyNKG2A-CD94 k_(a) k_(d) K_(D) K_(D) K_(D) ratio Analyte (1/Ms) (1/s) (nM) (nM) (cy/h) NKG2A.9 3.4 × 2.0 × 61 nM 1600 nM About 26 Fab 10⁵ 10⁻²

Example 4 Epitope Mapping of Anti-NKG2A Antibodies

Methods known in the art were used to probe regions of NKG2A to which anti-NKG2A antibodies, specifically, 13F3.A4 and NKG2A.9 antibodies, bind. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP) were used to probe binding epitopes of hNKG2A with the 13F3.A4 and NKG2A.9 antibodies.

HDX-MS experiments on mFc-hNKG2A-hCD94/NKG2A.9 provided 95% sequence coverage for mFc-NKG2A-CD94. FPOP measurements yielded 99% sequence coverage on NKG2A. The total sequence coverage for NKG2A was 100% with the combined HDX-MS and FPOP dataset. Similarly, HDX-MS experiments on mFc-hNKG2A-hCD94/13F3 provided 95% sequence coverage for mFc-NKG2A-CD94. FPOP measurements yielded 99% sequence coverage on NKG2A. The total sequence coverage for NKG2A was 100% with the combined HDX-MS and FPOP dataset.

As discussed in more detail in this Example, certain embodiments of the present invention relates to an anti-NKG2A monoclonal antibody or antigen-binding portion thereof that specifically binds to an epitope located within discontinuous regions spanning approximately the following amino acid residues as determined by HDX-MS and/or FPOP epitope mapping:

-   -   Region 1: ¹⁵⁵LSIDNEEEMKF¹⁶⁵ (amino acid residues 155 to 165 of         SEQ ID NO: 2 (native hNKG2A amino acid sequence);     -   Region 2: ¹⁷¹PSSWIGVFRNSSHHPW¹⁸⁶ (amino acid residues 171 to 186         of SEQ ID NO: 2);     -   Region 3: ¹⁹²LAFKHEIKDSDN²⁰³ (amino acid residues 192 to 203 of         SEQ ID NO: 2);     -   Region 4: L (amino acid residue 206 of SEQ ID NO: 2); and     -   Region 5: ²¹²QVNRLKSAQCGSSIIYHC²²⁹ (amino acid residues 212 to         229 of SEQ ID NO: 2).

In some embodiments, the invention is directed to an anti-NKG2A monoclonal antibody or antigen-binding portion thereof that specifically binds to an epitope located within discontinuous regions consisting of the following amino acid residues as determined by HDX-MS:

-   -   Region 1: ¹⁵⁵LSIDNEEEMKF¹⁶⁵ (amino acid residues 155 to 165 of         SEQ ID NO: 2);     -   Region 2: ¹⁷¹PSSWIGVFRNSSHHPW¹⁸⁶ (amino acid residues 171 to 186         of SEQ ID NO: 2);     -   Region 3: ¹⁹²LAFKHEIKDSDN²⁰³ (amino acid residues 192 to 203 of         SEQ ID NO: 2); and     -   Region 5: ²¹²QVNRLKSAQCGSSIIYHC²²⁹ (amino acid residues 212 to         229 of SEQ ID NO: 2).

Preparation for Epitope Mapping Experiments.

Prior to epitope mapping experiments, non-deuterated experiments were carried out to generate a list of common peptides for recombinant mFc-hNKG2A-hCD94 and protein complexes of mFc-NKG2A-CD94 and parental 13F3 Fab or NKG2A.9 Fab (15 μm 1:1 molar ratio). In the HDX-MS experiments, 5 μL of each sample (mFc-hNKG2A-hCD94 or mFc-hNKG2A-hCD94 with Fab) was diluted into 55 μL of deuterium oxide (D20) buffer (10 mM phosphate buffer, D20, pD 7.0) to start the labeling reactions. The reactions were carried out for different time periods: 20 seconds, 1 minute, 10 minutes and 4 hours. By the end of each labeling reaction period, the reaction was quenched by adding quenching buffer (100 mM phosphate buffer with 4 M guanidine hydrochloride (GdnCl) and 0.4 M tris(2-carboxylethyl)phosphine (TCEP), pH 2.5, 1:1, volume/volume), and 50 μL of quenched sample was injected into Waters HDX-MS system for analysis. The deuterium uptake levels of common peptic peptides were monitored in the absence or presence of Fabs.

Epitope Mapping of 13F3 and NKG2A.9 by HDX-MS.

HDX-MS.

HDX-MS probes protein conformation and conformational dynamics in solution by monitoring the rate and extent of deuterium exchange of backbone amide hydrogen atoms (R. Huang and G. Chen, Analytical and Bioanalytical Chemistry, 406: 6541-58 (2014); Wei et al., Drug Discovery Today, 19: 95-102 (2014). The level of HDX depends on the solvent accessibility of backbone amide hydrogen atoms and the protein hydrogen bonds. The mass increase of the protein upon HDX can be precisely measured by MS. When this technique is paired with enzymatic digestion, structure features at the peptide level can be resolved, enabling differentiation of surface-exposed peptides from those folded inside. Typically, the deuterium labeling and subsequent quenching experiments are performed followed by enzymatic digestion, peptide separation, and MS analysis.

FIG. 28A shows differential HDX of mFc-hNKG2A-hCD94 upon interaction with NKG2A.9 antibody. FIG. 28B shows differential HDX of mFc-NKG2A-CD94 upon interaction with 13F3.A4. The data in FIGS. 28A-B show that the epitopes are the same for NGK2A.9 and 13F.3.A4 antibodies Specifically, HDX-MS data analysis on the NKG2A9 antibody in mFc-NKG2A-CD94 indicated that NKG2A9 and the 13F3.A4 parental monoclonal antibody share the same epitope, which is the following four discontinuous regions of hNKG2A (residue numbers correspond to native hNKG2A sequence):

Region 1: ¹⁵⁵LSIDNEEEMKF¹⁶⁵ (amino acid residues 155 to 165 of SEQ ID NO: 2); Region 2: ¹⁷¹PSSWIGVFRNSSHHPW¹⁸⁶ (amino acid residues 171 to 186 of SEQ ID NO: 2); Region 3: ¹⁹²LAFKHEIKDSDN²⁰³ (amino acid residues 192 to 203 of SEQ ID NO: 2); and Region 5: ²¹²QVNRLKSAQCGSSIIYHC²²⁹ (amino acid residues 212 to 229 of SEQ ID NO: 2). Epitope Mapped to mFc-hNKG2A-hCD94 Sequence and Crystal Structure.

FPOP.

Fast photochemical oxidation of proteins (FPOP) is a protein footprinting technique to determine structural information by mapping oxidation induced by hydroxyl (OH) radicals. (Li et al., Analytical Chemistry, 2017, 89, 2250-2258). The extent of hydroxyl radical-induced oxidation depends directly on solvent accessibility of amino acids side chains and chemical properties of exposed amino acids. Hydroxyl radicals, produced from hydrogen peroxide (14202) via laser activation, are highly reactive and produce covalent irreversible modifications to the side chains without causing backbone cleavages. FPOP coupled with enzymatic digestion and MS analysis allows peptide level assessment of the changes in solvent accessibilities of side chains due to protein-protein interactions. Residue-level information can be achieved by gas-phase fragmentation of the selected peptide regions by MS/MS. This technique provides complementary information to HDX.

Epitope mapping by FPOP was performed on mFc-NKG2A-CD94 and mFc-NKG2A-CD94 complexed with parental 13F3 Fab (15 μM 1:1 molar ratio). Similarly, epitope mapping by FPOP was performed on mFc-NKG2A-CD94 and mFc-NKG2A-CD94 complexed with NKG2A.9 Fab (15 μM, 1:1 molar ratio).

For epitope mapping of both 13F3.A4 and NKG2A.9 antibodies, a krypton fluoride (KrF) excimer laser was used to generate hydroxyl radicals by the photolysis of H₂O₂, and the excitation wavelength was set as 248 nm to avoid any laser-induced conformation change of protein. Immediately before labeling, 5 μL of histidine and 5 μL of H₂O₂ were added to a protein aliquot. The final volume of protein solution was 50 μL, and the final concentrations of histidine was 500 μM and of H₂O₂ was 15 mM. The sample was then injected into fused silica tubing with an ultraviolet (UV) transparent window. The laser energy was adjusted to 70 mJ/pulse at a frequency of 7.4 Hz. Both FPOP and no laser control experiments were performed in triplicate. Each replicate was collected in a microcentrifuge tube containing 11 μL of quenching solution (50 nM of catalase and 20 mM of methionine). The samples were denatured, reduced, alkylated, deglycosylated, and digested with trypsin followed by liquid chromatography (LC)/MS analysis. The oxidation levels of tryptic peptides were monitored in the absence or presence of the NKG2A.9 Fab.

In FPOP experiments, four residues, M163, F179, H184, and L206, in hNKG2A exhibited significant reduction (P-value<0.025) in oxidation levels upon binding with parental 13F3 Fab. Similarly, in FPOP experiments, four residues, M163, F179, H184, and L206, in hNKG2A exhibited significant reduction (P-value<0.025) in oxidation levels upon binding with NKG2A9.

For both 13F3.A4 and NKG2A.9 antibodies, FPOP protection percentage upon binding of Fab was calculated as:

$\frac{\begin{matrix} {\left( {{Relative}\mspace{14mu} \% \mspace{14mu} {FPOP}\mspace{14mu} {difference}\mspace{14mu} {in}\mspace{14mu} {hNKG}\; 2A} \right) -} \\ \left( {{Relative}\mspace{14mu} \% \mspace{14mu} {FPOP}\mspace{14mu} {difference}\mspace{14mu} {in}\mspace{14mu} {hNKG}\; 2{A/{Fab}}\mspace{14mu} {complex}} \right) \end{matrix}}{{Relative}\mspace{14mu} \% \mspace{14mu} {FPOP}\mspace{14mu} {difference}\mspace{14mu} {in}\mspace{14mu} {hNKG}\; 2A} \times 100$

FIG. 29A displays protection percentages for four residues (M163, F179, H184, L206) in hNKG2A upon interaction with NKG2A.9. FIG. 29B displays FPOP protection percentages for four residues (M163, F179, H184, L206) in hNKG2A upon interaction with 13F3.A4. Epitope Mapped to mFc-NKG2A-CD94 Sequence and Crystal Structure.

As shown in FIG. 30, the epitopes of NKG2A.9 and 13F3.A4 antibodies determined by HDX-MS and by FPOP were mapped to the mFc-hNKG2A-hCD94 sequence. In the mFc-hNKG2A-hCD94 sequence, mFc is the mouse Fc region, which is linked via a linker sequence ASIEGR (SEQ ID NO: 123) (shown by a box with dashed lines) to the extracellular domain of hNKG2A, which is linked via a linker sequence GGSGGS (SEQ ID NO: 124) (shown by a box with dashed lines) to hCD94, which is human CD94 protein.

As shown in FIG. 31, the anti-NKG2A antibody epitope (for example, epitopes of NKG2A.9 and 13F3.A4 antibodies) can be visualized on the NKG2A/CD94/HLA-E crystal structure. Specifically, FIG. 31 depicts the anti-NKG2A antibody epitope (shown in black) on the NKG2A/CD94/HLA-E crystal structure (human CD94/NKG2A in complex with HLA-E crystal structure was published by Petrie et al., J. Exp. Med. 205: 725-35 (2008), which is incorporated by reference in its entirety.) The HLA-E crystal structure is indicated with a solid oval, and the CD94 crystal structure is indicated with a dotted oval.

In summary, based on the HDX-MS and FPOP experiments discussed herein, the NKG2A epitope to which the NKG2A.9 and 13F3.A4 antibodies bind includes the following discontinuous binding regions:

-   -   Region 1: LSIDNEEEMKF (amino acid residues 155 to 165 of SEQ ID         NO: 2);     -   Region 2: PSSWIGVFRNSSHHPW (amino acid residues 171 to 186 of         SEQ ID NO: 2);     -   Region 3: LAFKHEIKDSDN (amino acid residues 192 to 203 of SEQ ID         NO: 2);     -   Region 4: L (amino acid residue 206 of SEQ ID NO: 2); and     -   Region 5: QVNRLKSAQCGSSIIYHC (amino acid residues 212 to 229 of         SEQ ID NO: 2).

In other embodiments, based on the HDX-MS experiments discussed herein, the NKG2A epitope to which the NKG2A.9 and 13F3.A4 antibodies bind includes the following discontinuous binding regions:

-   -   Region 1: LSIDNEEEMKF (amino acid residues 155 to 165 of SEQ ID         NO: 2);     -   Region 2: PSSWIGVFRNSSHHPW (amino acid residues 171 to 186 of         SEQ ID NO: 2);     -   Region 3: LAFKHEIKDSDN (amino acid residues 192 to 203 of SEQ ID         NO: 2); and     -   Region 5: QVNRLKSAQCGSSIIYHC (amino acid residues 212 to 229 of         SEQ ID NO: 2).

Example 5 Epitope Binning Experiments

(1) P1-069366 Antibody Cross-Blocked 13F3.A4

An epitope binning experiment for P1-069366 and 13F3.A4 was performed by SPR using a “sandwich format”, where antibody 1 is immobilized onto a Biacore T100 CM5 chip, the recombinant human NKG2A-CD94 protein is flowed over the Biacore T100 CM5 chip and allowed to bind, and then antibody 2 is flowed over the chip. Specifically, 13F3.A4 or P1-antibodies were immobilized onto a CM5 sensor chip by amine coupling. A solution of recombinant NKG2A protein was flowed over the sensor chip where it was observed to bind both P1-069366 and 13F3.A4 antibodies. A solution of competing antibody was then flowed over the chip and additional binding was measured (indicative of co-binding to NKG2A). Finally, the sensor chip was regenerated using a solution of MgCl₂, which removed bound NKG2A and competing antibody. The experiment was conducted at 25° C. For analysis, non-specific binding to the reference flow cell was subtracted from binding to the analytical flow cells. P1-069366 antibody was directly immobilized to flow cell 2. Then, 200 nM NKG2A was flowered over flow cell 2, and approximately 600 RU was observed to bind. Subsequent injection of 13F3.A4 antibody, P1-069366 antibody, or buffer (HBS-P) did not result in increased binding signal. 13F3.A4 antibody was immobilized onto flow cell 3. Next, 200 nM NKG2A was flowed over flow cell 3, and approximately 900 RU was observed to bind. Subsequent injection of P1-069366 antibody, 13F3.A4 antibody, or buffer (HBS-P) did not result increased binding signal. In conclusion, P1-069366 antibody appeared to cross-block 13F3.A4 based on the results of the epitope binning experiment.

(2) 13F3.A4, 2G6.C2 Blocked Each Other as Well as HLA-E from Binding to Human NKG2A-CD94.

In another epitope binning and blocking experiment that was performed on an Octet HTX instrument using the “sandwich format”, 13F3.A4 antibody, 2G6.C2 antibody as well as HLA-E were tested for simultaneous binding to human NKG2A. The antibodies and biotinylated HLA-E were captured on separate Octet sensor tips. Next, the remaining capture sites were blocked if required, and human NKG2A-CD94 was bound. Subsequent binding of 13F3.A4 antibody, 2G6.C2 antibody, and HLA-E to pre-bound NKG2A-CD94 was tested, probing all pairwise combinations of these three samples. These epitope binning experiments showed that the 13F3.A4 and 2G6.C2 antibodies block each other as well as HLA-E from binding to human NKG2A-CD94.

(3) Binding Activity of 2G6.C2 Antibody

Octet experiments also showed binding of 2G6.C2 antibody not only to human NKG2A-CD94 but also to a cynomolgus NKG2A-CD94-mFc construct. The kinetics of 2G6.C2 antibody binding to human NKG2A-CD94 and NKG2C-CD94 heterodimers were measured on a Biacore T200 instrument at 37° C. by capturing the antibody on a CM4 chip with pre-immobilized anti-hFc antibody and flowing the antigens as analytes with an appropriate concentration range. All data were double-referenced and fitted to a 1:1 binding model using the Biacore T200 Evaluation Software version 3.1. The Biacore experiments showed that the 2G6.C2 antibody had a weaker binding affinity than the 13F3.A4 antibody and that it has some cross-reactivity to NKG2C, as summarized below.

Half-Life of Antibody/Analyte k_(a) k_(d) K_(D) Complex Captured Analyte (1/Ms) (1/s) (M) (seconds) 2G6.C2 hNKG2A- 4.0 × 3.6 × 9.1 × 19 CD94 10⁵ 10⁻² 10⁻⁸ 2G6.C2 hNKG2C- 2.8 × 3.0 × 1.1 × 23 CD94 10⁴ 10⁻² 10⁻⁶

Example 6 Discovery of Additional Anti-NKG2A Antibodies

As shown in FIGS. 32A-B, the heavy and light chain variable regions of certain anti-NKG2A antibodies (13F3.A4, NKG2A.9, and NKG2A.11) were aligned using Clutsal 2.1 software. This alignment resulted in the discovery of anti-NKG2A antibodies with the following consensus CDR sequences:

HCDR1: (SEQ ID NO: 10) SHSMN HCDR2: (SEQ ID NO: 11) AISSSSSYIYYADSVKG HCDR3: (SEQ ID NO: 12) EEWGLPFDY LCDR1: RASQGI S SALA (SEQ ID NO: 13, for NKG2A.9 antibody); RASQGI P SALA (SEQ ID NO: 154, for NKG2A.11 antibody); or RASQGI N SALA (SEQ ID NO: 155, for 13F3.A4 antibody). LCDR2: (SEQ ID NO: 14) DASSLKS LCDR3: (SEQ ID NO: 15) QQFNSYPLT

Example 7

Development of NKG2A.9 Antibody with an Inert Fc (IgG1.3) to Reduce or Prevent Fc Gamma Receptor (FcγR) Binding

Since NKG2A is an inhibitory receptor expressed on CD8+ T and NK cells, we believe that avoiding or reducing agonism or depletion of NKG2A⁺ CD8+ T or NK cells enhances anti-tumor immunity. Thus, blockade of the NKG2A/HLA-E interaction was desired with an anti-NKG2A antibody unable to interact with human FcγRs.

As summarized in Study 1 below, we assessed the role of the isotype in mediating tumor growth inhibition in the 1956 sarcoma model. NKG2A.2 antibody is a mouse surrogate antibody generated as an mIgG2a or mIgG1-D265A isotype to evaluate the role of Fc in antitumor activity.

Mouse Tumor Model Study 1

% Tumor Growth Inhibition Tumor Recipient Treatment (compared to Purpose Model Mice mAb(s) Regimen isotype control) Efficacy 1956 C57Bl/6 NKG2A.2 Day 0: 1 × 10⁶ At Day 21 of anti- sarcoma n = 10/grp (mIg2a) cells per mouse NKG2A.2-gl mNKG2A.2 NKG2A.2 subcutaneous D265A: 41% IgG2a (mIgG1- (SC) NKG2A.2-g2a: NKG2A.2 D265A) Days 6, 9, 12: 0% IgG1- Each isotype 10 D265A mg/kg isotypes intraperitoneal (IP)

At Day 21 post implant, NKG2A.2-mIGg1 D265A inhibited tumor growth by 41% compared to the isotype control. In other words, treatment of tumor-bearing mice with an anti-mNKG2A mAb containing an inert Fc (mNKG2A.2 mIgG1-D265A) led to potent antitumor activity. However, when NKG2A.2 mIgG2a was administered, the antibody did not inhibit tumor growth. In other words, when the NKG2A.2 antibody was tested with an Fc capable of interacting with mouse FcγRs (mNKG2A.2 mIgG2a), tumors grew faster in vivo.

When anti-hNKG2A mAb with an IgG4 Fc was tested, it inhibited NK cell responses, particularly in the presence of strong stimulation.

Collectively, these data showed the advantageous functions of inert Fc human IgG1.3 was the preferred isotype for the NKG2A.9 antibody.

Example 8 NKG2A.9 Antibody Enhanced the Functional Activity of T Cells

In this assay, the NKG2A.9 antibody was tested for its functional activity of reversing the inhibition of NK-κB signaling in a NKG2A-expressing Jurkat T cell line stimulated by CHO/scOKT3/HLA-E (Chinese Hamster Ovary cells that have been engineered to express the single chain OKT3 and the ligand, HLA-E). NKG2A-expressing Jurkat effector cells were co-cultured with CHO/scOKT3/HLA-E target cells at an effector cell-to target cell ratio (E:T) of 1:1. The NKG2A.9 antibody or isotype antibody (human IgG1.3) was added to the co-culture at titrating concentrations. Following four hours of stimulation at 37° C., luciferase activity was quantified using Bio-Glo Reagent (100 μl/well) and EnVision plate reader. Relative Luciferase Units (RLU) data was plotted using Prism v5.01 software from GraphPad Inc.

The NKG2A.9 antibody reversed the NKG2A/HLA-E mediated inhibition of T cell responses. Specifically, as shown in FIG. 33, the NKG2A.9 antibody reversed the inhibition of NK-κB signaling in a NKG2A-expressing Jurkat T cell line stimulated by CHO/scOKT3/HLA-E, with an EC₅₀ value of 0.2 nM.

Example 9

NKG2A.9 Antibody IFN-γ Production Alone and in Combination with Anti-PD-L1 Monoclonal Antibody

The NKG2A.9 antibody induced IFN-γ in NKG2A⁺ CD8 T isolated from healthy donor PBMC co-cultured with CHO/scOKT3/HLA-E/PD-L1 (Chinese Hamster Ovary cells that have been engineered to express the single chain OKT3 and the ligands HLA-E and PD-L1). Enhanced IFN-γ production is a measure of enhanced T-cell functionality. The effect was significantly amplified when NKG2A.9 was combined with anti-PD-L1 monoclonal antibody.

Briefly, as depicted in FIG. 34A, T cells isolated from healthy PBMCs were incubated with irradiated CHO/scOKT3/HLA-E/PD-L1 in the presence of NKG2A.9 antibody and an anti-PD-L1 antibody (BMS-936659), or a combination thereof for four days. The T cells were isolated from human whole blood using Ficoll gradient and the human T cell isolation kit (EasySep™ Human T Cell Isolation Kit, Stemcell Technologies). The cell viability was more than 90%, as determined by cell counter (Nexcelcom Cellometer Auto 2000). 1.5×10⁵ cells were co-cultured for four days with 2.5×10⁴ irradiated (67,000 RAD for 80 minutes; Rad Source Irradiator, RS-2000 Biological System) CHO/OKT3/HLA-E/PD-L1 cells in rhIL-15 (5 ng/ml) containing medium in the presence of either an isotype control antibody (human IgG1.3) or NKG2A.9 and/or anti-human PD-L1 antibody (BMS-936659). (CHO/scOKT3/HLAE/PD-L1 cells are Chinese Hamster Ovary cells that have been engineered to express the single chain OKT3 and the ligands HLA-E and PD-L1). At day four of the culture, cells were collected and stained with anti-human CD4, anti-human CD8, live/dead, anti-human NKG2A (27H4 clone). IFN-γ production was assessed by intracellular cytokine staining using the BD Cytofix/Cytoperm™ kit. Fixed cells were read on the BD LSRFortessa™. The percent of IFN-γ among NKG2A+ or NKG2A− CD8 T cells was calculated by FlowJo.

As shown in FIG. 34B, a dose-dependent enhancement of IFN-γ production by NKG2A⁺ CD8 T cells was observed when the NKG2A.9 antibody was combined with the anti-PD-L1 antibody. Notably, the increase in IFN-γ production was not observed on NKG2A⁻ CD8 T cells, which indicated that the effect of the NKG2A.9 antibody was specific and intrinsic to NKG2A⁺ CD8 T cells.

The NKG2A.9 antibody also induced IFN-γ in NKG2A+ CD8+ T cells isolated from human tumors co-cultured with CHO/scOKT3/HLA-E/PD-L1. The effect was significantly amplified when the NKG2A.9 antibody was combined with an anti-PD-L1 monoclonal antibody (BMS-936659). Briefly, as shown in in FIG. 35A-B, tumor-infiltrating lymphocytes (TIL) from renal cell carcinoma, melanoma, or endometrial tumor samples were co-cultured with irradiated CHO/scOKT3/HLA-E/PD-L1 in the presence of the NKG2A.9 antibody, anti-PD-L1, or their combination for four days. The NKG2A.9 antibody did not increase IFN-γ levels by NKG2A⁺ CD8 TIL over control monoclonal antibody, whereas anti-PD-L1 antibody stimulated IFN-γ levels by NKG2A⁺ CD8 TIL over isotype antibody (P=0.01). The greatest IFN-γ response was observed with the combination of the NKG2A.9 antibody and anti-PD-L1 mAb compared to isotype control antibody (P=0.003) as well as compared to anti-PD-L1 alone (P=0.04), as shown in FIG. 35B.

Example 10

NKG2A.9 Antibody was Desirably Internalized after Binding to NKG2A-Expressing Cell

As described in the Examples above, we observed down-regulation of NKG2A surface expression after incubation of human CD8+ T or NK cells with the NKG2A.9 antibody. To determine whether the NKG2A.9 antibody was internalized into the cell, Alexa Fluor 488-labeled NKG2A.9 antibody or isotype control monoclonal antibody (KLH human IgG1 antibody) was added to NKL cells, and the levels of intracellular and surface-bound monoclonal antibodies were measured. Internationalization of the antibody shows target engagement of the antibody to the receptor. As shown in FIG. 36A, the NKG2A.9 antibody demonstrated internalization within one hour and increased over 20 hours. The NKG2A.9 antibody was gradually internalized by NKL cells and reached a plateau level of about 40% internalization by 20 hours. To quantify internalization of the NKG2A.9 antibody in a dose-response format, the pH sensitive dye assay was performed using NKL cells. As shown in FIG. 36B, at two hours, the NKG2A.9 antibody demonstrated a dose-dependent internalization with an EC₅₀ of 0.5 nM.

Example 11 NKG2A.9 Antibody Increased Natural Killer Cell Degranulation and Lysis of HLA-E-Expressing Tumor Cells

In in vitro experiments using PBMCs from normal donors, anti-NKG2A antibody blockade of the NKG2A/HLA-E interaction among activated NK cells resulted in increased NK cell degranulation. As depicted in FIG. 37A, NK cells isolated from normal PBMC donors were activated overnight with recombinant human (rh) IL-2. NK cells were then co-cultured with the human B-lymphoblastoid target cell line 721.221 expressing HLA-E. As shown in FIG. 37B, the NKG2A.9 antibody, as compared to isotype, increased NK cell degranulation in a dose-dependent manner, as measured by % CD107a expression by flow cytometry. Reversal of NKG2A/HLA-E mediated inhibition of NK cell cytotoxicity was assessed in vitro. As depicted in FIG. 37C, HSC-3, a human oral cancer cell line, was selected based on its endogenous expression of HLA-E. NKL cells were co-cultured with the HSC-3 target cells labeled with Calcein AM (a cell-permanent dye) at a 20:1 effector to target ratio in the presence of NKG2A.9 or isotype control antibody (human IgG1.3). The amount of Calcein released after two hours was measured and used to indicate the level of target cell lysis. As shown in FIG. 37D, the NKG2A.9 antibody, as compared to isotype, increased lysis of HLA-E expressing tumor cells in a dose dependent manner.

Example 12 13F3.A4 Antibody Showed Enhanced NK Cell Functionality

FIG. 38A illustrates the method used to measure the effect of the 13F3.A4 antibody on IFN-γ production in NKL cells co-cultured with CHO/MICA/HLA-E. Similar to the NKG2A.9 antibody, FIG. 38B shows that the 13F3.A4 antibody increased IFN-γ production in natural killer lymphocytes (NKL) co-cultured with CHO/MICA/HLA-E (Chinese Hamster Ovary cells that have been engineered to express MICA and HLA-E) as compared to the isotype.

Example 13 Comparative Binding Affinities of NKG2A.9

A comparison of the kinetics and binding affinities of NKG2A.9, Z270, and monalizumab for binding to hNKG2A-hCD94 was made using a Biacore T200 biosensor instrument and a heterodimer of the extracellular domains of human NKG2A and CD94. The temperature was 37° C. and the running buffer was Hepes buffered saline (10 mM HEPES, 150 mM NaCl) at pH 7.4 supplemented with 0.05% Tween-20 and 1 g/L BSA. The antibodies were captured on a C1 sensor chip with pre-immobilized anti-human Fc capture reagent (Southern Biotech catalog number 2081-01). The extracellular domain of human NKG2A was flowed as analyte over the captured antibodies in two five-membered, three-fold concentration series with 250 nM top concentration: one concentration series was used for single-cycle kinetics and the other one for multi-cycle kinetics with a duplicate injection at 83 nM. All data were double-referenced and fitted to a 1:1 binding model using Biacore T200 Evaluation Software version 3.1. Two independent runs were performed, using a different flow cell for each monoclonal antibody in the second run. Thus, four K_(D) measurements were obtained. Where applicable, kinetic fit and steady state fit were both used to analyze the data.

FIG. 51A-D show the binding affinity of the NKG2A.9 antibody to human NKG2A-CD94 heterodimer at 37° C. as determined by Biacore in quadruplicate measurements using both single-cycle and multi-cycle kinetics. FIG. 51A-D show response units (RU) of NKG2A-CD94 binding versus time in seconds.

The NKG2A.9 antibody showed higher affinity (K_(D)=16 nM±0.4 nM; average±standard deviation) than Z270 (K_(D)=25 nM±3 nM) and monalizumab (K_(D)=26 nM 3 nM) in this assay, as summarized in the Table below. The overall affinity for NKG2A.9 is higher in this assay than in the experiment using NKG2A.9 Fab in Example 3; the difference may be due to a faster association rate (k_(a)), while the dissociation rate (k_(d)) is very similar. The faster association rate (k_(a)) in this assay may be reflective of by the more optimized conditions, in particular, the use of a flat sensor chip, which were used due to less reagent constraints for human versus cynomolgus NKG2A.

TABLE Comparative Affinity of Anti-NKG2A Antibodies and Half-Life of Antibody/Analyte Complex Half-life of Antibody/Analyte Captured k_(a) k_(d) K_(D) Complex Antibody Analyte (1/Ms) (1/s) (nM) (seconds, s) NKG2A.9 hNKG2A- 1.1 × 10⁶ ± 1.7 × 10⁻² ± 16.0 nM ±   37 to 45 s hCD94 0.1 × 10⁶ 0.2 × 10⁻² 0.4 nM Z270 8 × 10⁶ 2 × 10⁻¹ 25 nM ± 3 s to 4 s 3 nM Monalizumab 7 × 10⁶ 2 × 10⁻¹ 26 nM ± 3 s to 4 s 3 nM K_(D) was averaged from four measurements. Standard deviation is shown where application.

The half-life of a 1:1 complex, such as one antibody binding site bound to one NKG2A-CD94 heterodimer, is time that it takes for half of the amount of the antibody/analyte complex (here, the antibody:NKG2A-CD94 complex) to dissociate. The half-life can mathematically be described as ln(2)/k_(d). The half-life of the antibody:NKG2A-CD94 complex was approximately 12-fold longer for NKG2A.9 (41 seconds) than for both Z270 and monalizumab (3 to 4 seconds).

Under conditions of monovalent binding on a cell surface (e.g., due to low antigen expression), NKG2A.9 can thus engage NKG2A-CD94 complex for a longer time period than Z270 and monalizumab. We believe that this longer half-life of the antibody/NKG2A-CD94 complex allows increased efficiency in ligand blocking. In other words, the longer half-life of the NKG2A.9/NKG2A-CD94 complex compared to monalizumab/NKG2A-CD94 or Z270/NKG2A-CD94 provided evidence for the ability of the NKG2A.9 antibody to better block the NKG2A/HLA-E interaction compared to monalizumab or Z270.

Example 14 Properties of Anti-mNKG2A Antibodies In Vitro

Anti-mNKG2A surrogate antibodies, NKG2A.2 (20D5 clone, eBioscience, mIgG1-D265A) and NKG2A.3 (7E6 clone, mIgG1-D265A), had similar functional properties to the NKG2A.9 antibody when evaluated in vitro, allowing these antibodies to serve as surrogate antibodies for proof-of-concept, mechanism of action (MOA), and human dose projection studies. The anti-NKG2A mouse antibodies NKG2A.2 and NKG2A.3 had the following functional properties:

1) EC₅₀ values for binding to mNKG2A-expressing CHO cells were 0.2 nM (NKG2A.2) and 0.3 nM (NKG2A.3).

2) IC₅₀ values for blockade of Qa-1b tetramer binding to mNKG2A-expressing CHO cells were 0.3 nM (NKG2A.2) and 0.4 nM (NKG2A.3). (Qa-1b is the house homolog to HLA-E in humans. Thus, this is comparable to the CHO hNKG2A/HLA-E blocking assay).

3) NKG2A.2 and NKG2A.3 bound specifically to mNKG2A, as they did not bind to NKG2A^(−/−) NK or CD8 T cells.

4) NKG2A.2 and NKG2A.3 antibodies induced a dose-dependent increase in mouse NK cell degranulation.

Example 15

Anti-mNKG2A Antibodies, Both Alone and in Combination with Other Agents, Induced Anti-Tumor Activity In Vivo

(1) Anti-mNKG2A Antibodies Induced Antitumor Activity as a Monotherapy in Mouse Models

Anti-mNKG2A monoclonal antibodies (including the NKG2A.2 and NKG2A.3 antibodies) when administered as a single agent inhibited tumor growth in CT26 colon carcinoma, 1956 sarcoma, and A20 B cell lymphoma subcutaneous (SC) models as summarized in Study 2 below.

Summary of Mouse Tumor Model Studies 2-4

% Tumor Growth Study Tumor Recipient Treatment Inhibition (compared No. Purpose Model Mice mAb(s) Regimen to isotype control) 2 Dose CT26 BALB/c NKG2A.3 Day 0: 1 × 10⁶ cells At Day 20 titration colon n = 10/ (mIgG1-D265A) per mouse SC NKG2A.3 (10 mg/kg) + of anti- carcinoma group Anti-PD-1 Ab Days 6, 10, 13, 17, Anti-PD-1 Ab: 96% mNKG2A.3 (mIGg1 D265A) 20: NKG2A.3 (1 mg/kg) + NKG2A 10, 3 , 1, Anti-PD-1 Ab: 96% 0.3 mg/kg Anti-PD-1 Ab: 88% intraperitoneal (IP) NKG2A.3(3 mg/kg) + PD-1 (mPD1-4h2- Anti-PD-1 Ab: 76% mg1-D265A, NKG2A.3(0.3 mg/kg) + mouse surrogate Anti-PD-1 Ab: 71% for nivolumab) 10 NKG2A.3 (3 mg/kg): 56% mg/kg IP NKG2A.3(10 mg/kg): 49% Each antibody NKG2A.3(1 mg/kg): 31% administered at 10 NKG2A.3(0.3 mg/kg): 0% mg/kg IP 3 Efficacy CT26 BALB/c NKG2A.3 Day 0: 1 × 10⁶ cells At Day 24 of anti- colon n = 10/ (mIgG1-D265A) per mouse SC NKG2A.3 + anti-PD-1 Ab mNKG2A.3 in carcinoma group Anti-PD-1 Ab Days 6, 10, 13,17, (Clone 4H2, mIgG1- combination (mIGg1 D265A) 20: D265aA: 91% with other Anti-TIGIT Ab Each antibody NKG2A.3 + anti-TIGIT checkpoint (mIg2a) administered at 10 Ab (Clone 4H2, mIGg1- inhibitors mg/kg IP D265A: 88% NKG2A.3: 42% Anti-PD-1 Ab: 61% Anti-TIGIT Ab: 78% 4 Efficacy A20 B cell BALB/c NKG2A.2 Day 0: 5 × 10⁶ cells At Day 23 of anti- lymphoma n = 10/ (mIgG1-D265A) per mouse SC NKG2A.3 + Anti-PD-1 Ab: mNKG2A.2 in group Anti-PD-1 Ab Days 6, 9, 12, 15, 94% combination with (mIgG1-D265A) 18: Anti-PD-1 Ab: 75% checkpoint Anti-TIGIT Ab Each antibodies NKG2A.3: 58% inhibitors in (mIg2a) dosed at 10 mg/kg NKG2A.3 + Anti-TIGIT subcutaneous IP Ab: 51% Anti-TIGIT Ab: 32% Tumor-Free Mice at End of Study Anti-NKG2A A + Anti- PD-1 Ab: 8/9 tumor-free mice at the end of the study (TF) Anti-NKG2A Ab + Anti- TIGIT Ab: 4/9 TF Anti-PD-1 Ab: 3/9 TF Anti-NKG2A Ab: 1/9 TF Anti-TIGIT Ab: 0/9 TF Isotype: 0/9 TF As shown in FIG. 39, in a dose titration study (Study No. 2 described above) with the CT26 tumor model, the NKG2A.3 antibody reduced tumor size when administered at 10 mg/kg, 3 mg/kg, and 1 mg/kg doses (48%, 56%, and 30% reduction in mean tumor volume, respectively).

(2) Combination of Anti-mPD-1 and Anti-mNKG2A Antibodies Enhanced Anti-Tumor Activity Compared to Either Single Agent Alone in Multiple Mouse Tumor Models.

Co-blockade with the anti-NKG2A (mNKG2A.3-mG1-D265A (7E6 clone) and anti-PD-1 antibodies (PD1-4H2-mG1-D265A, 6A1 clone) reduced tumor growth in models where anti-NKG2A monoclonal antibody alone showed single-agent activity. The combination of anti-mPD-1 and anti-mNKG2A antibodies demonstrated greater anti-tumor activity than either single agent alone in the CT26 tumor model.

In the CT26 model, single-agent treatment with anti-mNKG2A or anti-mPD-1 antibodies reduced the mean tumor volume by 43% and 61%, respectively. Co-blockade of NKG2A and PD-1 led to a relatively stronger inhibition of tumor growth, with 7/10 mice tumor-free (TF) at the end of the study (FIG. 40D) and a 91% decrease in mean tumor volume (FIG. 40E). FIG. 40A-D show the tumor volume at various time points post tumor implantation in mice (n=10/group) treated with isotype (FIG. 40A), anti-mNKG2A antibody alone (FIG. 40B), anti-mPD-1 antibody alone (FIG. 40C), or a combination of anti-mNKG2A and anti-mPD1 antibodies (FIG. 40D). FIG. 40E shows the average tumor volume as a function of time (days post tumor implantation) in mice treated with isotype, anti-mNKG2A antibody alone, anti-mPD-1 antibody alone, or a combination of anti-mNKG2A and anti-mPD-1 antibodies.

Similar effects were observed in mice inoculated with 1956 sarcoma or A20 lymphoma SC tumors, as summarized in Studies 3 and 4 below.

Summary of Mouse Tumor Model Studies 3 and 4

% Tumor Growth Study Tumor Recipient Treatment Inhibition (compared No. Purpose Model Mice mAb(s) Regimen to isotype control) 3 Efficacy CT26 BALB/c NKG2A.3 Day 0: 1 × 10⁶ cells At Day 24 of anti- colon n = 10/ (mIgG1-D265A) per mouse SC NKG2A3 + anti-PD-1 mNKG2A.3 in carcinoma group Anti-PD-1 Ab Days 6, 10, 13, Ab: 91% combination (mIGg1 D265A) 17, 20: NKG2A.3 + anti- with other Anti-TIGIT Ab Each antibody TIGIT Ab (10A7 checkpoint (mIg2a) administered at 10 clone, mIgG1-D265A): inhibitors mg/kg IP 88% NKG2A.3: 42% Anti-PD-1 Ab: 61% Anti-TIGIT Ab: 78% 4 Efficacy A20 B cell BALB/c NKG2A.2 Day 0: 5 × 10⁶ cells NKG2A.3 + anti-PD-1 of anti- lymphoma n = 10/ (mIgG1-D265A) per mouse SC Ab: 94% mNKG2A.2 in group Anti-PD-1 Days 6, 9, 12, 15, Anti-PD-1 Ab: 75% combination with (mIgG1-D265A) 18: NKG2A.3: 58% checkpoint Ab Each antibodies NKG2A.3 + anti-TIGIT inhibitors in Anti-TIGIT dosed at 10 mg/kg Ab: 51% subcutaneous (mIg2a) Ab IP Anti-TIGIT Ab: 32% lymphoma model Tumor-Free Mice at End of Study NKG2A + anti-PD-1 Ab: 8/9 tumor-free mice at the end of the study (TF) NKG2A + Anti-TIGIT Ab: 4/9 TF Anti-PD-1 Ab: 3/9 TF NKG2A: 1/9 TF Anti-TIGIT Ab: 0/9 TF Isotype: 0/9 TF

NKG2A/PD-1 co-blockade improved tumor growth inhibition even in models where anti-NKG2A monoclonal antibodies alone (NKG2A.3-mG1-D265A (7E6 clone) or NKG2A.2-mg1-D265A (4F12 clone)) did not show single-agent activity. In the M109 lung tumor model, co-blockade of anti-NKG2A and anti-PD-1 antibodies led to a 72% decrease in the mean tumor volume compared to isotype control, as summarized in Study 5 below.

Summary of Mouse Tumor Model Study 5

% Tumor Growth Study Tumor Recipient Treatment Inhibition (compared No. Purpose Model Mice mAb(s) Regimen to isotype control) 5 Efficacy M109 lung BALB/c NKG2A.2 Day 0: 2 × l0⁵ cells At Day 25 of anti- carcinoma n = 10/ (mIgG1-D265A) per mouse SC NKG2A + Anti-PD-1 mNKG2A.2 in group Anti-PD-1 Ab Day 7, 10, 13: Ab: 72% combination (mIgG1-D265A) Each antibody Anti-PD-1 Ab: 17% with anti- administered at 10 Anti-NKG2A Ab: mPD-1 mg/kg IP 11%

In the BR5.1 ovarian tumor model, co-blockade of NKG2A and PD-1 led to a 36% decrease in the mean tumor volume compared to isotype control, as summarized in Study 6 below.

Summary of Mouse Tumor Model Study 6

% Tumor Growth Study Tumor Recipient Inhibition (compared No. Purpose Model Mice mAb(s) Treatment Regimen to isotype control) 6 Efficacy BR5.1 FVB/NJ NKG2A.2 Day 0: Fragmented At Day 26 of anti- ovarian n = 15/ (mIGg1- tumors SC NKG2A + PD-1: mNKG2A.2 in group D265A) Days 5, 8, 11, 14, 17: 36% combination PD-1 (mIGg1- NKG2A.2 10 mg/kg NKG2A: 5% with anti- D265A) IP PD-1: 0% mPD-1 PD-1 5 mg/kg IP

We did not observe activity of anti-mNKG2A alone or in combination with anti-mPD-1 in the MC38 colon carcinoma model, as shown in Study 7 below.

Summary of Mouse Tumor Model Study 7

% Tumor Growth Study Tumor Recipient Treatment Inhibition (compared No. Purpose Model Mice mAb(s) Regimen to isotype control) 7 Efficacy of MC38 colon C57Bl/6 NKG2A.3 Day 0: 1e6 cells No efficacy anti- adeno- n = 10/ (mIGg1- per mouse SC observed mNKG2A.3 with carcinoma group D265A) Days 6, 8: anti-mPD-1 PD-1 (mIGg1- Each antibody D265A) administered at 10 mg/kg IP

(3) Anti-NKG2A and Anti-PD-1 Antibodies Enhanced Antitumor Activity and Functionality of Mouse Natural Killer (NK) Cells and CD8 Tumor Infiltrating Lymphocytes (TILs).

NKG2A/PD-1 co-blockade efficacy was dependent on both CD8+ T and NK cells. CT26-bearing mice were depleted of either CD8+ T or NK cells prior to treatment with anti-mNKG2A and anti-mPD-1 monoclonal antibodies. Mice treated with anti-mNKG2A (NKG2A.3, 7E6 clone, mIgG1-D265A)/anti-mPD-1 (4H2 clone, mIgG1-D265A) antibodies had an increase in mean tumor volume when depleted of either CD8+ T cells (191% increase, mean tumor volume 2271±699 mm³) or NK cells (94% increase, mean tumor volume 1509±800 mm³) compared to mice that did not receive depleting monoclonal antibodies (mean tumor volume 778±686 mm³), as summarized in Study 8 below.

Summary of Mouse Tumor Model Study 8

% Tumor Growth Study Tumor Recipient Treatment Inhibition (compared No. Purpose Model Mice mAb(s) Regimen to isotype control) 8 Depletion CT26 colon BALB/c NKG2A.3 Day 0: 1e6 cells At Day 21 of NK carcinoma n = 10/ (mIGg1- per mouse SC NKG2A3 + PD-1: and group D265A) Days 5, 12, 19: 60% CD8 T- PD-1 (mIGg1- anti-asialo GM1 NKG2A3 + PD-1 + cells D265A) (50 μg/mouse) or anti-asialo GM1: Anti-asialo anti-CD8a (200 μg/ 22% GM1 (e- mouse) IP NKG2A3 + PD-1 + Bioscience) Days 6, 10, 13, 17, anti-CD8: 0% Anti-CD8a 20: (53.6.7, Each antibody BioXCell) administered at 10 mg/kg IP

NKG2A/PD-1 co-blockade enhanced functionality of NK and CD8+ T TIL. After four doses of monoclonal antibodies (Day 12 post treatment initiation), NK and CD8+ T TILs were analyzed by flow cytometry. Anti-mNKG2A/anti-mPD-1 treatment led to an increased frequency of CD107a⁺ NK cells relative to isotype control-treated groups, as shown in FIG. 41A. Immuno-phenotyping studies in the CT26 model showed that co-blockade with the anti-NKG2A and anti-PD-1 antibodies showed a statistically significant increase in NK and tumor specific CD8+ T-cell cytotoxicity and IFN-γ. Anti-mNKG2A/anti-mPD-1 treatment led to an increased frequency of CD107a⁺ NK cells with an approximately 1.5-fold increase relative to isotype control-treated group. A similar increase in the frequency of cytotoxic CD8+ T cells was seen among tumor-specific AH-1⁺ CD8+ T cells, as shown in FIG. 41B with an approximately 13-fold increase in CD107a+ Granzyme B+ AH1+ CD8 T cells relative to isotype control-treated group. NKG2A/PD-1 co-blockade also increased the frequency of IFN-γ producing AH-1⁺ CD8+ T cells with an approximately 5.5-fold increase in IFN-γ+ AH-1⁺ CD8+ T cells relative to isotype control-treated group, as shown in FIG. 41C.

In summary, anti-NKG2A and anti-PD-1 antibodies, either alone or in combination, increased the frequency of cytotoxic CD8+ T cells among tumor-specific AH-1⁺ CD8 T cells. The administration of anti-NKG2A and anti-PD-1 antibodies increased NK and tumor-specific CD8 T cell cytotoxicity and IFN-γ in the CT model.

(4) Anti-mPD-1 Antibodies Inhibited Tumor Growth to a Greater Extent when Combined with Anti-mNKG2A Compared to Combination with Anti-mLAG-3 or Anti-mTIGIT Antibodies in a Mouse Model of Sarcoma.

It was previously shown that dual blockade of PD-1 with anti-LAG-3 and anti-PD-1 antibodies with anti-TIGIT antibodies synergistically delayed tumor growth as compared with single agent treatment in mouse tumor models. (Woo et al., “Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape.” Cancer Res 72:917-27 (2012); Johnston R J et al., “The immunoreceptor TIGIT regulates antitumor and antiviral CD8 (+) T cell effector function.” Cancer Cell 26:923-937 (2014).

We examined the effect of combining anti-mPD-1 antibody with either anti-mNKG2A (NKG2A.3, 7E6 clone, mIgG1-D265a), anti-mLAG-3 (1A5 clone, mIgG1-D265A), or anti-mTIGIT (10A7 clone, mIgG1-D265A) antibodies on tumor growth control in the 1956 sarcoma model, as summarized in Study 9 below.

Summary of Mouse Tumor Model Study 9

% Tumor Growth Inhibition Study Tumor Recipient Treatment (compared to isotype No. Purpose Model Mice mAb(s) Regimen control) 9 Efficacy 1956 C57Bl/6 NKG2A.3 Day 0: 1 × 10⁶ cells At Day 27 of anti- sarcoma n = 10/ (mIGg1-D265A) per mouse SC NKG2A.3 + Anti-PD-1 mPD-1 in group Anti-PD-1 Ab Days 6, 9, 12, 15, Ab: 97% combination (mIGg1-D265A) 18: Anti-PD-1 Ab + with anti- TTGIT Each antibody Anti-LAG-3 Ab: 91% mNKG2A.3 (mIGg1-D265A) administered at 10 Anti-PD-1 Ab: 77% and other Anti-LAG-3 Ab mg/kg IP Anti PD-1 Ab + checkpoint (mIGg1-D265A) Anti-TIGIT Ab: 72% inhibitors NKG2A.3-D265A: 24% Anti-LAG-3-D265A Ab: 0% Anti-TIGIT-D265A Ab: 0% Tumor-Free Mice at End of Study (TF) NKG2A + PD-1: 6/10 TF LAG-3 + PD-1: 4/10 TF PD-1: 2/10 TF PD-1 + TIGIT: 1/10 TF NKG2A: 1/10 TF LAG-3: 0/10 TF TIGIT: 0/10 TF Isotype: 0/10 TF

PD-1/NKG2A dual blockade showed the strongest anti-tumor efficacy (6/10 tumor-free mice) compared to the combination of 1) antiPD-1/anti-LAG-3 antibodies (4/10 tumor-free mice) and 2) anti-PD-1/anti-TIGIT antibodies (1/10 tumor-free mice).

Anti-mNKG2A antibodies was combined with other checkpoint inhibitors including anti-mCTLA-4 and anti-LAG-3 antibodies to enhance antitumor efficacy in several mouse tumor models. In the 1956 model, anti-mNKG2A antibody demonstrated strong activity when combined with anti-mCTLA-4, with 87% reduction in mean tumor growth volume and 3/10 mice tumor-free compared to isotype control, as described in studies summarized in Study 10 and FIG. 42A-E.

Summary of Mouse Tumor Model Study 10

% Tumor Growth mAb(s) (with Inhibition Study Tumor Recipient isotype in Treatment (compared to isotype No. Purpose Model Mice parenthesis) Regimen control) 10 Efficacy of 1956 C57Bl/6 NKG2A.3 Day 0: 1 × 10⁶ At Day 22 anti- sarcoma n = 10/ (mIGg1- cells per mouse NKG2A.3 + Anti- mNKG2A.3 in group D265A) SC CTLA-4 Ab: 86% combination Anti-PD-1 Ab Days 6, 9, 12: NKG2A.3 + Anti-PD-1 with lower (mIGg1- NKG2A: 10 Ab: 76% doses of D265A) mg/kg IP Anti-CTLA-4 mIgG2a other Anti-CTLA-4 Anti-PD-1 Ab: Ab: 51% checkpoint Ab (mIgG2a) 0.3 mg/kg IP NKG2A.3: 26% inhibitors Anti-CTLA-4 Anti-PD-1 mIGg1 Ab: 0.1 mg/kg IP D265A Ab: 25%

FIG. 42A-E shows the anti-tumor activity of anti-mNKG2A (10 mg/kg) and anti-mCTLA-4 (0.1 mg/kg) antibodies, either alone or in combination, in the 1956 mouse sarcoma model. FIG. 42A-D show the tumor volume at various time points post tumor implantation in mice treated with isotype (FIG. 42A), anti-mCTLA-4 antibody (FIG. 42B, CTLA-4 IgG2a, 0.1 mg/kg), anti-mNKG2A antibody (FIG. 42C, 10 mg/kg), or combination of anti-mNKG2A and anti-mCTLA-4 (FIG. 42D). FIG. 42E shows the average tumor volume as a function of time (days post tumor implantation) in mice treated with isotype, anti-mCTLA-4 alone, anti-mNKG2A alone, or combination of anti-mNKG2A and anti-mCTLA-4.

In the A20 lymphoma intravenous model, anti-NKG2A antibody alone had a survival benefit, with a 10% survival rate. Combination therapy with either anti-mPD-1 or anti-mLAG-3 antibodies extended the survival benefit to 50% and 70%, respectively. The triple combination of anti-mNKG2A, anti-mPD-1, and anti-mLAG-3 antibodies provided the greatest benefit with an 80% survival rate as summarized in Study 11 below and shown in FIG. 43.

Summary of Mouse Tumor Model Study 11

% Tumor Growth mAb(s)(with Inhibition Study Tumor Recipient isotype in Treatment (compared to isotype No. Purpose Model Mice parenthesis) Regimen control) 11 Survival A20 B BALB/c NKG2A.3 Day 0: 2 × 10⁵ cells % Survival on Day 60: following cell n = 10/ (mIGg1-D265A) per mouse IV Anti-NKG2A + Anti- treatment lymphoma group Anti-PD-1 Ab Days 4, 7, 10, 13, PD-1 + Anti-LAG-3 with anti- (mIGg1-D265A) 16: Abs: 80%, P = <0.0001 mNKG2A.3 in Anto-LAG-3 Each antibody Anti-NKG2A + Anti- combination with (mIGg1-D265A) administered at 10 LAG-3 Abs: 70%, checkpoint inhibitors mg/kg IP P = 0.0004 in IV Anti-NKG2A + Anti- lymphoma PD-1 Abs: 50%, model P = 0.006 Anti-LAG-3 Ab: 40%, P = 0.001 Anti-PD-1 Ab: 20%, P = <0.0001 Anti-NKG2A Ab: 10%, P = 0.028 (P value compared to isotype control)

FIG. 43 shows the anti-tumor activity of anti-mNKG2A, anti-PD-1, anti-LAG3 antibodies, both alone and in combination in the A20 systemic lymphoma model. Specifically, FIG. 43 shows the percent survival at various time points post tumor implantation in mice (n=10/group) treated with isotype, anti-mNKG2A, anti-mPD-1, anti-LAG3 alone, or a combination thereof.

Taken together, these data showed that combinatorial blockade of anti-NKG2A, anti-PD-1, anti-CTLA-4, and anti-LAG-3 antibodies has enhanced efficacy in reducing mouse tumor growth.

(5) Anti-mNKG2A Monoclonal Antibody Administered In Vivo Downregulated Surface NKG2A on Mouse NK Cells

After treatment with anti-mNKG2A monoclonal antibody (NKG2A.3 (7E6 clone), mIgG1-D265A), the NKG2A expression level was reduced on both splenic and TIL NK cells as compared with the isotype, as shown in FIG. 44. FIG. 44 shows the percent of NKG2A-expressing NK cells among NKG2A+ NK cells as measured on day five after first treatment in spleen and tumor in a murine CT26 colon carcinoma model. We observed downregulation of the NKG2A expression after administration of the anti-NKG2A antibody, which indicated that it can be used as a marker of target engagement. Downregulation refers to the downregulation of the surface NKG2A expression. Target engagement means that the anti-NKG2A antibody engages with the NKG2A receptor.

Together with the internalization data discussed herein, these data support that NKG2A downregulation will serve as a target engagement marker for the NKG2A.9 antibody.

Example 16 Biophysical Properties, Affinity and Epitope Mapping, and Immunogenicity

As described herein, the variable sequence of fully human anti-NKG2A monoclonal antibody NKG2A.9 is derived from the 13F3.A4 hybridoma with a VH-I107T germline reversion mutation and a VK-N30S mutation to mitigate the deamidation liability of the VK-N30 amino acid residue. The constant region was derived from an IgG1f backbone (IgG1.3) and includes three mutations on the heavy chain: L234A, L235E, and G237A to minimize or eliminate antibody binding to Fcγ receptors and C1q. The NKG2A.9 antibody is also known as 13F3-VH-I107T-VH-N305 IgGf1.3 and NKG2A.9 IgGf1.3. The Table below summarizes the biophysical characteristics of the NKG2A.9 antibody based on the analysis of the material derived from the ExpiCHO transient expression system.

Property Method Results Identity SDS-PAGE (sodium dodecyl Heavy and light chain bands confirmed sulfate-polyacryamide gel Deglycosylated molecular weight (MW) = 143,683 electrophoresis) Dalton (Da) (as predicted) LC-MS/MS peptide map *Heavy Chain (HC): observed 48,893 Da, inferred 48,900 Da, predicted 48,900 Da *Light Chain (LC) = observed 23,207 Da, inferred 23,211 Da, predicted 23,210 Da >99% sequence and disulfide structure confirmed by peptide mapping and mass spectrometry. Isoform and N-297 glycosylation on heavy chain were confirmed. Purity/Homo CE-SDS (capillary Non-reduced (NR): 97.0% monomer, impurities geneity electrophoresis sodium include 2.1% HHL (heavy chain-heavy chain-light dodecyl sulfate) chain), 0.4% HL (heavy chain-light chain), 0.1% HH SEC (Size Exclusion (heavy chain-heavy chain), 0.3% Chromatography) Reduced (R): LC 29.5%, HC 69.8%, NGHC 0.7% SE-MALS (combination of 98.1% monomer Size Exclusion 95.2%(146 kDa), 2.63% (505 kDa), 1.6% (109 kDa) Chromatography with Multi- 86.4% main peak at 35.5 minutes, 13.6% pre-main Angle Light Scattering peak. analysis) GOF (78%), G1F (17%), G2F (1%), Man5 (4%), HIC-HPLC (Hydrophobic Main peak pI = 8.9 (59%), Acidic species 35%, Interaction Chromatography- Basic species 7%; pI range 8.6-9.1 High Performance Liquid Chromatography) CE (Capillary electrophoresis, Glycans) cIEF (Capillary isoelectric focusing) Chemical LC-MS/MS (Liquid Very low Modifications Chromatography-Tandem mass spectrometry) peptide map Affinity Biacore K_(D) = 36 nM (1:1 binding model); k_(a) (1/Ms) = human 3.0 × 10⁵, k_(d) (1/s) = NKG2A** 1.1 × 10⁻²at 37° C.; cross-reacts with cynomolgus NKG2A Thermal DSC (Differential scanning Tm1 = 68° C., Tm2 = 75° C., Tm3 = 83° C. Stability colorimeter) (diluted into Reversibility at 68° C. = 92%, at 75° C. = 49% and storage buffer) Reversibility *Deglycosylation and partial reduction method used for light and heavy chain mass confirmation. ** Human NKG2A-CD94 heterodimer (hNKG2A-CD94-mFc) used as antigen for binding studies.

The biophysical properties of the NKG2A.9 antibody were favorable. The identity of the NKG2A.9 antibody was confirmed by SDS-PAGE and mass spectrometry analysis. The purity of the antibody was greater than 98% as tested by size exclusion chromatography (SEC). A single N-glycosylation site was confirmed at N297 on the heavy chain, with a glycan profile that was consistent with the glycan profile of IgG1 monoclonal antibodies expressed in CHO cells. Only fucosylated glycans (G0f, G1f, G2f, and Man5) were found on them. The NKG2A.9 antibody had good thermal stability and reversibility, meaning that the antibody retained its structural integrity under thermal stress and had modest refolding properties when stress was released.

Example 17 Preclinical Pharmacokinetics Studies

The pharmacokinetics data supported efficacy of the NKG2A.9 antibody. After intravenous (IV) administration to cynomolgus monkeys, the NKG2A.9 exhibited linear antibody pharmacokinetic (PK) characteristics at doses ≥0.5 mg/kg, with a predicted human half-life (T½) of 16 days. The human efficacious dose targeting the steady-state trough concentration (C_(trough,ss)) for achieving complete (99%) receptor occupancy (RO) in blood was projected to be 2.5 mg/kg IV, every four weeks. The dose after subcutaneous (SC) administration may vary depending on SC bioavailability in humans (typically 50-100%) and whether a slow SC absorption rate improves the C_(trough,ss).

Formulation efforts showed that a platform formulation approach can be used for a ready-to-use (RTU) formulation. Preliminary viscosity and stability evaluation of the NKG2A.9 antibody at high (150 mg/mL) concentration and the predicted human efficacious dose (2.5 mg/kg) indicate that subcutaneous administration may be possible under optimized formulation conditions.

The toxicology data showed that the NKG2A.9 antibody can be safely administered. Doses of 0 mg/kg, 0.5 mg/kg, 10 mg/kg, and 50 mg/kg in cynomolgus monkeys were well tolerated with no abnormalities identified. Receptor occupancy at Day 43 (36%, 84%, and 97% at 0.1 mg/kg, 10 mg/kg, and 50 mg/kg, respectively) and target engagement (downregulation of surface NKG2A) were demonstrated at all doses. Downregulation refers to the downregulation of the surface NKG2A expression. We observed downregulation of the NKG2A expression following administration of the antibody, which indicated that it can be used as a marker of target engagement (i.e. anti-NKG2A antibody engages with the NKG2A receptor).

Example 18 Pharmacokinetics of Anti-mNKG2A.3 in Mice

Pharmacokinetic studies were conducted with an anti-mNKG2A mouse surrogate (NKG2A.3) that has similar properties to the NKG2A.9 antibody following intravenous (IV) or intraperitoneal (IP) administration to C57BL6 mice. Table 3 summarizes the pharmacokinetic parameters obtained from the studies.

TABLE 3 Pharmacokinetic Parameters of an Anti-mNKG2A Mouse Surrogate (NKG2A.3) after IV and IP Administration to C57BL6 Mice (Mean ± Standard Deviation (SD), n = 4 or 5)* Route of Dose Cmax Tmax AUCtot T½ CLT Vss administration (mg/kg) (nM) (hours) (μM × day) (days) (mL/day/kg) (mL/kg) IV 0.3  24 ± 6.8 0.25 ± 0** 0.075 ± 0.010 3.2 ± 0.2 26 ± 3.5 121 ± 16 (non-tumor- 1  79 ± 7.8 0.25 ± 0**  0.38 ± 0.036 3.9 ± 0.5 17 ± 1.7 108 ± 15 bearing) 10 940 ± 327 0.25 ± 0** 7.3 ± 2.0 9.4 ± 2.5 9.7 ± 2.5  128 ± 19 IP 0.1 6.9 ± 0.8  2 ± 0 0.014 ± 0.004 n.a. n.a. n.a. (tumor- 10 993 ± 87   2 ± 0 8.0 ± 1.1 n.a. n.a. n.a. bearing) Note: PK parameters were obtained using non-compartmental analysis of serum drug concentration-time data. n.a. stands for not applicable. *Serum drug levels were obtained from serial micro-sampling and corrected for either a theoretical dilution factor of 17 or an experimentally determined dilution factor of 20; **First sampling time point.

The NKG2A.3 antibody exhibited nonlinear pharmacokinetics in mice. With an intravenous dose ratio of 1:3:30, the Area Under the Curve (AUC) ratio was 1:5.1:97. The terminal half-life increased from 3.2 days to 9.4 days when the doses increased from 0.3 mg/kg to 10 mg/kg. Similarly, with an IP dose ratio of 1:100, the AUC ratio was 1:571 between 0.1 and 10 mg/kg. In addition, the formation of anti-drug antibodies (ADAs) did not affect the PK of NKG2A.3 significantly. Collectively, these data showed that the NKG2A.3 antibody underwent target-mediated drug disposition (TMDD) in mice.

The IP bioavailability was complete when comparing the AUC values at 10 mg/kg between IV and IP routes. In addition, there was no apparent difference in the systemic exposure between tumor- and non-tumor-bearing mice as well as between C57BL6 and BALB/c mice.

In addition, a single-dose PK/pharmacodynamic (PD) study was conducted with the NKG2A.3 antibody in the CT26 model, where the time course of drug levels and receptor occupancy (RO) was determined in both the circulation and tumors. The average tumor-to-serum drug concentration ratio was 0.10±0.07 (N=33), with the RO in blood and tumor infiltrating lymphocytes (TILs) being complete at the mouse efficacious dose of 1 mg/kg.

Example 19 Pharmacokinetics of the NKG2A.9 Antibody in Cynomolgus Monkeys

We characterized the pharmacokinetic characteristics of the NKG2A.9 antibody in cynomolgus monkeys at a dose range of 0.5 mg-50 mg/kg). The PK parameters of the NKG2A.9 antibody obtained from a single-dose toxicokinetic (TK)/PD and tolerability study are summarized in Table 4 below.

TABLE 4 Pharmacokinetic Parameters of the NKG2A.9 Antibody after IV Administration to Cynomolgus Monkeys (Mean ± SD, N = 3) Dose Cmax Tmax AUC0-42 d T½ CLT Vss (mg/kg) (nM) (hour) (μM × day) (day) (mL/day/kg) (mL/kg) 0.5 0.11 ± 0.01 0.5 ± 0*   1 ± 0.2 14 ± 6 3.1 ± 0.9 53 ± 7 10  2.5 ± 0.29 0.5 ± 0* 22 ± 4  18 ± 3** 2.4 ± 0.5 57 ± 6 50 9.8 ± 2.5 0.5 ± 0* 101 ± 18 23 ± 4 2.5 ± 0.5  77 ± 12 Note: PK parameters were obtained using non-compartmental analysis of serum drug concentration-time data. *First sampling time point; **Two drug concentrations affected by ADAs were not included for T½ calculation.

The AUC_(0-42d) was dose-proportional between 0.5 mg/kg and 50 mg/kg IV doses, with half-life ranging from 14 days to 23 days. In addition, the PK results were affected by ADAs only in one monkey. Furthermore, PK/PD modeling of the monkey blood RO data revealed that the serum drug EC₅₀ was 20±5.4 nM, which is in agreement with the in vitro EC₅₀ of 31±5.4 nM.

In conclusion, the studies demonstrated that the anti-NKG2A antibody showed good receptor occupancy and was well tolerated in cynomolgus monkeys.

Example 20 Human Pharmacokinetics of the NKG2A.9 Antibody

The human PK of the NKG2A.9 antibody is assumed to be same as that in monkeys. As a result, the predicted human intravenous dose of the NKG2A.9 antibody is 10 mg/kg, and the predicted half-life of the NKG2A.9 antibody in humans is 16 days, as summarized in Table 5 below.

TABLE 5 Predicted Human Pharmacokinetic Parameters of the NKG2A.9 Antibody IV Dose AUC_(tot) T½ CLT V_(SS) (mg/kg) (μM × day) (day) (mL/day/kg) (mL/kg) 10 24 16 2.7 61

Example 21 Projection of Human Efficacious Dose

The human efficacious dose of the NKG2A.9 antibody was projected using an RO-based approach. The available in vitro and in vivo preclinical data, including RO in mice and monkeys as well as mouse antitumor efficacy, were evaluated. The maximum antitumor efficacy in combination with anti-mPD-1 was demonstrated in 1956 and CT26 syngeneic models at a dosage regimen of 1 mg/kg administered every three to four days for a total of five doses (Q3/4D×5), with no further improvement in efficacy at doses up to 10 mg/kg. Importantly, at 1 mg/kg, the NKG2A receptors in both blood and tumors (i.e., TIL) were fully occupied, indicating complete RO. In addition, PK/PD modeling of monkey blood RO data revealed agreement between the in vitro and in vivo RO EC₅₀, which suggested that the in vitro EC₅₀ can be used to predict the in vivo RO. Taken together, the human efficacious dose of the NKG2A.9 antibody is projected by targeting 99% RO in blood at trough, using an in vitro human RO EC₅₀ of 1 nM. Because the average tumor-to-serum drug concentration ratio observed with NKG2A.3 in mice was about 0.10, along with the fact that a similar ratio was observed with an anti-OX40 antibody (BMS-986178) in human tumor biopsies samples, targeting 99% RO in blood at trough would lead to about 90% RO in tumors throughout the treatment period. Consequently, the projected human efficacious dose is 2.5 mg/kg administered every four weeks. At this dose, the predicted human steady-state AUC and C_(trough) cover the exposure achieved at the maximum efficacious dose of 1 mg/kg Q3/4Dx5 in mice.

Example 22 Stability and Formulation of the NKG2A.9 Antibody

Formulation efforts have shown that a platform formulation approach can be used for the development of a ready-to-use (RTU) formulation for the NKG2A.9 antibody. Preliminary viscosity and stability evaluation at 150 mg/mL indicated that the NKG2A.9 antibody can be administered via subcutaneous administration under optimized formulation conditions.

A formulation evaluation including a freeze-thaw stability study and an accelerated stability study have been performed for the NKG2A.9 antibody at a concentration of 25 mg/mL, as summarized in Table 6 below.

TABLE 6 Stability of the NKG2A.9 Antibody at 25 mg/mL in Platform Formulation Property Method(s) Results Freeze/Thaw (F/T) UV, SEC No F/T stability risk revealed (1 hour at −80° C., 1 hour at room temperature (RT) × 5) Solubility/Concentration Profile UV, SEC At least 25 mg/mL in platform buffer (20 mM histidine, pH 6.0, 260 mM sucrose, 0.05 mM DTPA, 0.05% polysorbate 80) Accelerated Stability UV, SEC, cIEF, LC- 3 months at 40° C. = 5% increase 25 mg/mL 3 months at 4° C., 25° C., and MS/MS peptide in LMW* 40° C. in platform formulation mapping, functional 3 months at 40° C. = 4% increase in (flow cytometry) HMW* 3 months at 40° C. = 12%/month increase in acidic variant 3 months at 40° C. = 18% increase in deamidation of N326 in VSNK region (SEQ ID NO: 162) *LMW = Low Molecular Weight, HMW = High Molecular Weight

No physical stability issues were observed during freeze-thaw stress (five cycles) at 25 mg/mL in platform formulation (20 mM histidine buffer pH 6.0, 260 mM sucrose, 50 μM pentetic acid, 0.05% polysorbate 80). Accelerated stability studies in platform buffer at 25 mg/mL were conducted at 4° C., 25° C., and 40° C. Over three months under the conditions tested, minor chemical modifications that did not affect functional activity were observed in the CDR region of the NKG2A.9 antibody. These minor chemical modifications in the NKG2A.9 antibody included changes in oxidation, deamidation, and isomerization of amino acid residues that are expected due to storage of the solution in, for example, an elevated temperature, did not adversely affect the binding activity of the antibody. Deamidation of N326 in the VSNK region (SEQ ID NO: 162) of the Fc was observed under stressed conditions (about 18% after three months at 40° C.), which is typical for IgG1 monoclonal antibodies. After storage at 25° C. for three months, deamidation was observed to be acceptable at about 1.4%. No physical stability issues or loss of functional activity were observed over three months under tested conditions.

No issues with viscosity (apparent viscosity=7.3 centipoise (cP)) or aggregation (0.4% soluble aggregates) were observed upon concentration of the NKG2A.9 antibody to 150 mg/mL. No physical stability issues were observed during freeze-thaw stress (five cycles) at 150 mg/mL in platform formulation. The NKG2A.9 antibody at 150 mg/mL in platform formulation was subjected to accelerated stability for three months at 4° C., 25° C., and 40° C., as summarized in Table 7 below.

TABLE 7 Stability of the NKG2A.9 Antibody at 150 mg/mL in Platform Formulation Property Method(s) Results Solubility/Concentration Profile UV, SEC, Viscosity At least 150 mg/mL in platform buffer (20 mM (viscometer) histidine, pH 6.0, 260 mM sucrose, 0.05 mM DTPA, 0.05% polysorbate 80) Apparent viscosity = 7.3 ± 0.1 CP Accelerated Stability UV, SEC 3 months at 4° C. = 0.1% increase in LMW 150 mg/mL 3 months at 4° C., 3 months at 4° C. = 0.8% increase in HMW 25° C. and 40° C. in platform 1 month at 25° C. = 0.4% increase in LMW formulation 1 month at 25° C. = 0.8% increase in HMW Significant aggregation and clipping observed at 25° C. and 40° C. beyond 1 month.

Minor physical stability issues were observed at 4° C. after three months and at 25° C. after one month on stability (0.8% increase in high molecular weight aggregates, HMW, at both temperatures). Clipping and physical instability were observed at elevated temperatures of 25° C. and 40° C. after storage for more than one month. The instability seen after storage for longer time at elevated temperature could potentially be explained by a slight contamination of the discovery lot used for the evaluation.

Example 23 Tissue Distribution of the NKG2A.9 Antibody

Immunohistochemistry using the NKG2A.9 Antibody on a panel of 20 frozen human tissues showed occasional mononuclear cell expression, but no unexpected binding. There was strong staining in small subsets of mononuclear cells, mainly in the spleen, and rare staining of mononuclear cells in tonsil, uterus, stomach, small intestines, thymus, and liver.

Example 24 Single-Dose Pharmacokinetics, Toxicokinetics, Tolerability, and Pharmacodynamics Study of NKG2A.9 in Cynomolgus Monkeys

Cynomolgus monkey is the preclinical toxicology species because of similar binding of the whole antibody to NKG2A. In addition, immunohistochemistry analysis of monkey tissues, using the NKG2A.9 antibody, is similar to that of humans, showing positive staining in small subsets of mononuclear cells. As discussed herein, the NKG2A.9 antibody binds to both NKG2A and NKG2C in monkeys, whereas it selectively binds to NKG2A and not NKG2C in humans. Since NKG2C is an activating receptor, it is possible that in monkeys there could be simultaneous upregulation of immune responses (due to NKG2A blockade) and downregulation of immune responses (due to NKG2C blockade).

The NKG2A.9 antibody was administered as a single intravenous dose to cynomolgus monkeys (n=3 per group, mixed sex) at 0 mg/kg, 0.5 mg/kg, 10 mg/kg, and 50 mg/kg, and animals were observed for 42 days. Study endpoints included clinical observations, body weight, clinical pathology, pharmacokinetics and toxicokinetics, anti-drug antibody (ADA) formation, RO, surface NKG2A expression, and PBMC immunophenotyping. All animals were immunized with simian immunodeficiency virus (SIV) Gag and Nef, expressed in two separate adenovirus-5 vectors, to evaluate enhancement of immune responses as a potential PD endpoint (SIV-reactive T cell [tetramer] assay on Day 22; ex vivo T cell response to antigen on Day 22). This was a non-terminal study, without necropsy or histopathology.

All doses were well tolerated at dosages up to 50 mg/kg (AUC_(0-42days)≤101 μM·day). There were no NKG2A.9 antibody-related clinical signs, changes in body weight, or NKG2A.9 antibody-related changes in hematology or clinical chemistry, as summarized in Table 8 below.

TABLE 8 Summary of Results for the NKG2A.9 Antibody: Single-dose IV TK, Tolerability, and PD in Monkeys (n = 3/group) Study Endpoint NKG2A.9 Antibody-Related Results Clinical signs None Body weight No significant changes Hematology, clinical No significant changes chemistry Receptor occupancy Day 1, 4 hours: 81%, 99%, and 100% at 0.5 mg/kg, 10 mg/kg, and 50 mg/kg, respectively Day 43: 36%, 84%, and 97% at 0.5 mg/kg, 10 mg/kg, and 50 mg/kg, respectively Downregulation of surface Day 1 (4 hours post-dosing): Surface expression was 50%, 52%, and NKG2A (target engagement 58% (relative to pre-dose) at 0.5 mg/kg, 10 mg/kg, and 50 mg/kg marker) Day 43: Surface expression was 65-80%, 47-107%, and 41-51% at 0.5 mg/kg, 10 mg/kg, and 50 mg/kg, respectively Response to immunization No change in ex vivo recall response to SIV Gag or Nef. An apparent NKG2A.9-mediated trend of decrease in the percentages of Nef- and Gag-specific (tetramer+) CD8+ cells. Within the antigen-specific CD8+ T cell populations, there was an apparent trend of decrease in the percentage of T_(EM) cells. Toxicokinetics At 50 mg/kg, the Cmax was 9.8 μM on Day 1, 0.5 h, and the AUC_(0-42 days) was 101 μM · day Anti-drug antibody (ADA) ADA observed in one animal (10 mg/kg) beginning at the first time point, Day 8

ADA was observed in one animal (10 mg/kg) beginning at the first time point, Day 8. Pharmacokinetics/toxicokinetics were linear in the dose range evaluated. At the high dosage of 50 mg/kg, the Cmax was 9.8 μM (on Day 1, 0.5 h) and the AUC_(0-42days) was 101 μM·day. In summary, the single-dose monkey study showed RO, downregulation of surface expression of NKG2A as a marker of target engagement, and acceptable TK without any adverse findings for a single dose administered at dosages up to 50 mg/kg (AUC_(0-42days)≤101 μM·day).

Example 25 Cytokine Release Assays

An in vitro cytokine release assay was performed using soluble NKG2A.9 antibody and whole blood from 15 human donors. Cytokines and chemokines were evaluated by a Luminex panel of 75 cytokines/chemokines to determine if treatment with the NKG2A.9 antibody poses a safety risk of cytokine release syndrome. No NKG2A.9-related induction of the human cytokines or chemokines assayed was observed.

Example 26 Immunogenicity Risk Assessment for Anti-NKG2A Antibodies

Therapeutic antibodies have the potential to elicit an immune response in patients against the therapeutic antibody. This immune response is usually manifested by the generation of anti-drug antibodies (ADA), such as through a human anti-human antibody (HAHA) response, which can alter exposure, neutralize therapeutic function, and/or even result in serious clinical consequences for the patient. The “foreignness” of the therapeutic antibody, which is recognized as “non-self” by the immune system, is thought to be the main driver of an MHC class II-mediated ADA immune response. It is important for a safe and effective anti-NKG2A antibody as described herein to have low or no immunogenicity. The potential for human immunogenicity of several anti-NKG2A monoclonal antibodies was evaluated by in silico HLA binding tools and in vitro by DC:T cell proliferation assays. As discussed herein, the NKG2A.9 antibody was engineered to have low human immunogenicity liabilities as assessed by in silico HLA binding tools. The risk of various anti-NKG2A antibodies (NKG2A.6, NKG2A.9, and NKG2A.11 antibodies) to elicit an undesirable immune response in humans against the antibodies was determined to be low based on in vitro DC:T cell proliferation assays.

1. In Silico HLA Binding Analysis was Used to Engineer Anti-NKG2A Antibodies with Low Immunogenicity Risk

T cell activation and proliferation are required steps to develop an immune response. Specifically, HLA Class II binding of peptide antigen is a critical step in developing high affinity IgG antibody mediated immune response. Using an in silico HLA binding tool (IEDB) (See, e.g., Wang P, et al., PLoS Comput Biol. 4(4) (2008)), we analyzed the CDR regions of monoclonal antibodies by dividing the amino acid sequence into overlapping 15 peptides and ranking the binding for 27 HLA DRB1 alleles (which covers about 95% of the human population) against all other peptides submitted to IEDB database. Peptides that rank in the top 10 percentile for a given HLA allele are considered a “binder,” and a region that shows a binder for 50% of the alleles is designated as a “binding cluster.” A “binder” or “binding clusters” within a biologic molecule are considered to be a driving factor in the development of immunogenicity (Sinu, P et al., Clinical & Developmental Immunology Vol. 2013 (2013)).

As shown in FIG. 54A, the 13F3.A4 antibody contains two HLA binding clusters; multiple peptides spanning the VH CDR 2 region and the VL CDR2 region were predicted to bind to up to 70% or 19/27 HLA call II DRB1 alleles (as denoted by the shaded numbers under the sequence in FIG. 54A). As discussed herein, a “binding cluster” is a region that shows a binder for 50% of the alleles; a shaded number 6 means that 60% of the alleles show binding and is, therefore, a “binding cluster.”

In addition, as shown in FIG. 54B and described herein, a VH-I107T framework reversion (that is, substituting isoleucine with threonine at position 107) removed a binding cluster from the 13F3.A4 antibody. This 1107T substitution of the 13F3.A4 antibody was selected, such as isoleucine at position 107 was selected in the NKG2A.9 antibody (T a position 107 is shown by the arrow in the second row of FIG. 54B).

As shown in FIG. 54C and described herein, to engineer and minimize immunogenicity potential of the 13F3.A4 antibody, in the light chain CDR1 of the 13F3.A4 antibody, the asparagine (N) at the 30 position (first row of FIG. 54C) was engineered to a proline (P) (second row of FIG. 54c ), glutamine (Q) (third row of FIG. 54C) or serine (S) (fourth row of FIG. 54C). The N30S substitution (as shown in the fourth row of FIG. 54C) was selected because it showed no HLA binding potential (as indicated by “0” in the shaded region). This N30S substitution of the 13F3.A4 antibody (N at position 30) was selected for inclusion in the NKG2A.9 antibody (S at position 30). In FIG. 54B-C, only certain portions of the sequences of the 13F3.A4 are shown for clarity.

2. Immunogenicity Risk of Anti-NKG2A Antibodies (NKG2A.6, NKG2A.9, and NKG2A.11) was Low Based on In Vitro DC:T Cell Proliferation Assay Results

After an HLA Class II molecule binds a peptide antigen, the next critical step in developing an immune response to a therapeutic antibody is the activation of CD4+ T cells. This T cell activation occurs as a result of the recognition of a cognate peptide-MHC complex (HLA) on an antigen presenting cell. T cell activation and proliferation (amongst multiple other factors) are required to develop an immune response. In vitro, peripheral blood mononuclear cell (PBMC)-based immunogenicity prediction assays using diverse donor sets are used to determine whether a molecule is potentially immunogenic by its ability to stimulate these specific, activated CD4+ T cells ex vivo.

An in vitro DC:T cell proliferation assay (see, e.g., methods described in Joubert M K, et al., PLOS ONE 11(8) (2016)) was conducted for several anti-human NKG2A antibodies (NKG2A.6, NKG2A.9, and NKG2A.11 antibodies) to further assess the human immunogenicity potential of these antibodies. Briefly, PBMCs from healthy volunteers were isolated by Ficoll (GE Healthcare). Gradient centrifugation and human lymphocyte antigen (HLA) Class II were characterized using polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes (ProImmune). A panel of 40 PBMC donors composed of HLA Class II types closely matching the world population frequencies was used for an assay run.

Monocytes isolated from PBMC using a negative bead based method (Intellicyt) were cultured in DC Media (Lonza) containing 11-4 and GM-CSF to generate immature dendritic cells (DC), pulsed with anti-human NKG2A antibodies (NKG2A.6, NKG2A.9, and NKG2A.11 antibodies) and quality control proteins for four hours followed by a two-day DC maturation step in media containing TNF-a, IL-1b, IL-6, and PGE2.

Pulsed DC were added to autologous PBMC-labeled with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) to monitor proliferation and plated in 96-well plates in six replicates at 200,000 cells per well in DC media (Lonzo) containing pen-strep (Gibco) for seven days, after which media was washed away and cells were labeled with an anti-human CD4 APC (BD Biosciences) monoclonal antibodies. After removal of the unbound anti-CD4 monoclonal antibody cells with a wash step, cells were fixed with 3.7% formalin (Sigma) in PBS analyzed by flow cytometry to determine the percentage of proliferating CD4+ T cells.

As shown in FIG. 55, NKG2A.9 produced CD4+ T cell proliferation response compared to media pulsed dendritic cells in only three out of 40 donors (7.5%) in this in vitro DC:T cell assay. This result was comparable to the 12.5% CD4+ proliferation for the control monoclonal antibody (bevacizumab), which has been demonstrated to have a very low immunogenicity in the clinic (“low control”) (Saffari F., et al., Int J Cancer Manag.; 11(11)(2018)). The “high control” antibody (ATR-107) showed CD4+ proliferation in 70% of donors, and has been shown to have a high ADA rate of 76% in clinical studies (Hua, F., et al., The Journal of Clinical Pharmacology, 54: 14-22 (2014)).

NKG2A.11 also produced CD4+ proliferation response in only 3 out of 40 donors. The NKG2A.6 antibody did not produce CD4+ proliferative response in any donor. Repeatability data for the high and low control antibodies for this assay show a Coefficient of Variation % (CV %) of 5-10%. Therefore, none of these monoclonal antibodies are significantly differently in their low immunogenicity risk. To summarize, the risk of the NKG2A.6, NKG2A.9, and NKG2A.11 antibodies to elicit an undesirable immune response in humans against the antibodies was determined to be low based on in vitro DC:T cell proliferation assays.

Example 27 Single-Dose Intravenous Toxicokinetic, Tolerability, and Pharmacodynamics Study in Monkeys

The NKG2A.9 antibody was well tolerated when given intravenously as a single dose to cynomolgus monkeys at 0.5 mg/kg, 10 mg/kg, and 50 mg/kg (AUC_(0-42days) 101 μM·day), and no abnormalities were identified by clinical pathology.

Specifically, this study was conducted to determine the potential toxicity of the NKG2A.9 antibody when given intravenously as a single dose to monkeys (1) to determine the systemic exposure to NKG2A.9, and (2) to assess the pharmacodynamic response to NKG2A.9, after immunization with adenovirus serotype 5 (Ad5) vectors expressing simian immunodeficiency virus proteins gag and nef (Ad5-gag and Ad5-nef, respectively). The NKG2A.9 antibody was administered intravenously as a slow bolus injection at doses of 0 mg/kg (vehicle), 0.5 mg/kg, 10 mg/kg, or 50 mg/kg to groups of three protein-naive monkeys (1 or 2 per sex). Doses were administered at 0.25 mL/kg, 0.5 mL/kg, or 2.5 mL/kg in a vehicle/carrier of 20 mM histidine, 260 mM sucrose, pH 6.0. The 2 Ad5 vectors were administered intramuscularly in the posterior quadriceps or caudal thighs in 1 injection with approximately 3.3 □10⁹ viral particles at 0.5 mL. Criteria for evaluation included survival, toxicokinetics, clinical observations, body weights, clinical pathology evaluations, and immunotoxicological and pharmacodynamic assessments (anti-drug antibodies, peripheral blood lymphocyte phenotyping, natural killer and T-cell activation, ex vivo recall responses to Nef and Gag, antigen-specific T-cell phenotyping, and NKG2A receptor occupancy and receptor expression). The monkeys were observed throughout the six-week post-dose period, after which they were returned to stock.

The NKG2A.9 antibody AUC(0-T) and Cmax exposures increased in a dose proportional manner at the dose levels evaluated. After intravenous administration, the systemic exposures in males were comparable to those in females at all dose levels. Because there were no substantial sex differences in systemic exposure values, all results and conclusions were based upon sex-combined data. One out of nine monkeys given the NKG2A.9 antibody at 10 mg/kg developed anti-drug antibodies, and the same monkey affected by the anti-drug antibodies exhibited a more rapid decline in drug concentrations compared to other monkeys.

The toxicokinetic summary for the NKG2A.9 antibody is shown below:

Toxicokinetic Summary - Mean Sex-combined Values NKG2A.9 Antibody Dose Parameter 0.5 mg/kg 10 mg/kg 50 mg/kg Cmax; □g/mL 16.8 380 1,470 AUC(0-1008 h); 3,620 79,000 365,000 All animals survived and were returned to stock at the end of the study. The NGK2A.9 antibody was well tolerated at all doses with no effects on clinical observations, body weight, or clinical pathology. There were no NKG2A.9-mediated changes in percentages of peripheral blood T cells, helper T cells, cytotoxic T cells, B cells, NK cells, activated CD4⁺, CD8⁺, effector memory CD8⁺ T cells, or NK cell enriched lymphocytes. Ex vivo recall responses to gag and nef peptides were also not altered. Percentages of antigen-specific CD8⁺ T cells were not altered, although there was high variability across all doses.

At four hours following dosing on Day 1, group mean NKG2A receptor occupancy on NK cell enriched lymphocytes was 81%, 99%, and 100% relative to pretest at 0.5 mg/kg, 10 mg/kg, and 50 mg/kg NKG2A.9, respectively. By Day 43, for monkeys dosed with 0.5 mg/kg NKG2A.9, receptor occupancy decreased to a range from 21% to 45%; for monkeys dosed with 10 mg/kg NKG2A.9, receptor occupancy decreased to a range from 72% to 95%. By 4 hours after dosing on Day 1, group mean NKG2A receptor expression on NK cell enriched lymphocytes was 50%, 52%, and 58% relative to pretest at 0.5 mg/kg, 10 mg/kg, and 50 mg/kg NKG2A.9, respectively, which showed internalization or downregulation of the receptor following dosing with NKG2A.9. By Day 43, NKG2A receptor expression ranged from 65% to 80%, 47% to 99%, and 41% to 46% at 0.5 mg/kg, 10 mg/kg, and 50 mg/kg NKG2A.9 antibody, respectively.

In conclusion, the NKG2A.9 antibody was well tolerated by monkeys following a single intravenous administration at ≤50 mg/kg (mean sex-combined AUC[0-1008h]≤365,000_(u)g·h/mL). Receptor occupancy and reduced receptor expression were observed at all doses tested.

Example 28 One-Month Intermittent Dose Subcutaneous and Intravenous Toxicity Study in Cynomolgus Monkeys

A one-month study was conducted to determine the potential toxicity of the NKG2A.9 antibody when administered to cynomolgus monkeys once weekly for one month (1) to evaluate the potential reversibility of any findings, (2) to determine systemic exposure to NKG2A.9, and (3) to provide data to support use of NKG2A.9 in humans. NKG2A.9 was administered by subcutaneous injection at doses of 0 mg/kg (vehicle), 10 mg/kg or 100 mg/kg to three groups of five monkeys/sex/group. Formulations for the subcutaneous doses of 10 mg/kg or 100 mg/kg NKG2A.9 included 2000 U/mL rHuPH20. In addition, NKG2A.9 was administered by intravenous injection (slow bolus) at a dose of 1 mg/kg to a single group of five monkeys/sex. All doses were administered at 2 mL/kg in a vehicle of 20 mM histidine, 250 mM sucrose, 0.05 mM diethylenetriamine pentaacetic acid (DTPA), and 0.05% (w/v) polysorbate-80 (PS-80), pH 6.

Criteria for evaluation included survival, toxicokinetics, clinical observations, body weights, visual food consumption estimates, physical (including respiratory and neurologic) and ophthalmologic examinations, clinical pathology evaluations, immunophenotyping, natural killer (NK) cell activation, receptor expression (RE), receptor occupancy (RO), organ weights, and gross and microscopic pathology analyses. Scheduled necropsies were conducted after 1 month of weekly dosing—5 doses total—(3/sex/group) and following an 8-week recovery period (2/sex/group).

On Day 22, mean NKG2A.9 systemic exposures (AUC[0-168h]) increased dose proportionally between 10 mg/kg and 100 mg/kg. There were no substantial sex differences at any dose. Accumulation of NKG2A.9 was observed following repeated administration at all dose levels, with mean systemic exposures (AUC[0-168h]) that were approximately 2.5-fold to 2.9-fold those on Day 1. The presence oftreatment-emergent anti-drug/NKG-2A.9 antibodies were detected in 1 of 10, 2 of 10, and 3 of 10 monkeys at 1 mg/kg (intravenous), 10 mg/kg (subcutaneous), or 100 (subcutaneous) mg/kg, respectively, on and/or after Day 8; these had no substantial impact on mean systemic exposures to NKG2A.9.

The toxicokinetic summary for the NKG2A.9 antibody is presented in the following table:

NKG2A.9 Dose 10 mg/kg 100 mg/kg 1 mg/kg (Subcutaneous with (Subcutaneous Parameter^(a) Period (Intravenous) 2000 U/mL rHuPH20) with 2000 U/mL Cmax Day 1 29.4 143 1.270 (μg/mL) Day 22 49.3/49.9 416/372 3,120/3,100 AUC(0-168 h) Day 1 1,960 19,200 170,000 (μg · h/mL) Day 22 4,820/4,850 55,500/52,700 425,000/424.000 ^(a)Values were calculated with data from all available monkeys/only monkeys without detectable treatment-emergent anti-NKG2A.9 antibodies (ADAs), which were detected on and/or after Day 8 (i.e., 168 hours after dosing on Day 1).

There were no NKG2A.9-related mortalities; all monkeys survived to their scheduled necropsies. NKG2A.9 was well tolerated at all doses with no effects on clinical observations, body weight, visual food consumption estimates, cardiovascular, neurological, ophthalmologic, clinical pathology, or histopathologic parameters,

There were no NKG2A.9-related changes in T cell (total T, helper T, or cytotoxic T), B cell, or NK cell numbers or activation of NK cell-enriched peripheral blood cell populations.

Dose-dependent receptor occupancy (RO) on NK cells was observed, (84%, 95%, and 100% at 1 mg/kg, 10 mg/kg, and 100 mg/kg NKG2A.9, respectively), four hours after the first dose. Thereafter, at all timepoints through Day 29, the group mean RO range was 72% to 84% and 94% to 98% at 1 mg/kg and 10 mg/kg, respectively, with maximal (100%) RO at 100 mg/kg. While RO was complete and sustained throughout the recovery phase (through Day 85) at 100 mg/kg, partial loss of RO was observed at 1 mg/kg and 10 mg/kg. At 1 mg/kg, partial loss of RO was observed from Day 71 through Day 85 (Group mean RO of 39%), which correlated with a drop in serum NKG2A.9 concentrations below 4 μg/mL. At 10 mg/kg, partial loss of occupancy was observed on Day 85 (Group mean RO of 74%), which correlated with a drop in serum NKG2A.9 concentrations below 50 μg/mL.

Engagement of NKG2 by NKG2A.9 is known to result in partial downregulation as a consequence of receptor internalization. There was a non-dose-dependent reduction of NKG2 RE on NK cells during the dosing phase (overall range of 36 to 75% of pretest values across all doses) with minimal recovery during the recovery phase at 1 mg/kg, and no recovery at 10 mg/kg or 100 mg/kg.

In conclusion, NKGA.9 was well tolerated by cynomolgus monkeys for one month at ≤100 mg/kg/week (mean AUC≤425,000 μg·h/mL) with evidence of robust target engagement at all doses. NKG2A.9-mediated in vivo blockade of NK cell NKG2A/HLA-E interaction in cynomolgus monkeys did not induce immune cell activation, suggesting the lack of, or minimal contribution of the NKG2A pathway in the context of a disease-free, physiological setting. Based on the tolerability and lack of anatomic or clinical pathology findings, the no-observed-adverse-effect level (NOAEL) was considered to be 100 mg/kg, which is also considered the highest non severely toxic dose (HNSTD).

Example 29 Immunohistochemical Analysis of NKG2A Expression in Tumors

We believe that the activity of the NKG2A.9 antibody reduces the inhibitory activity of NKG2A and leads to enhanced cytotoxicity at the tumor site. Consequently, criteria for tumor prioritization includes the expression of NKG2A and HLA-E.

To identify potential tumor indications, preliminary profiling of NKG2A in selected tumor types was assessed with FITC-conjugated NKG2A.6 (the parent clone of the NKG2A.9 antibody) on frozen sections. NKG2A was present in a small fraction of mononuclear cells and primarily distributed in the tumor stroma, as shown in FIG. 45. FIG. 45 shows representative examples of NKG2A staining in commercially procured human tumor samples stained with FITC-conjugated NKG2A.6 antibody and negative control reagent (anti-KLH human IgG1.1-FITC). In FIG. 45, “Cervical” indicates cervical carcinoma; “head & neck”” indicates head and neck squamous cell carcinoma; “bladder” indicates bladder carcinoma; and NSCLC-Adc indicates non-small cell lung cancer, adenocarcinoma. “St” are tumor stroma, and “TuEp” are tumor epithelial cells.

Image analysis of NKG2A expression by immunohistochemistry on frozen tumor sections was performed. “Hot spot” image analysis using HALO™ software was performed on three spots per sample. Staining with isotype control was subtracted from staining with the FITC-conjugated NKG2A.6 antibody. This image analysis revealed a relatively high abundance of NKG2A expression in cervical and head and neck carcinomas, moderate expression in bladder and non-small cell lung carcinomas, and low expression in pancreatic and colorectal carcinomas, as shown in FIG. 46. FIG. 46 shows NKG2A expression levels across different tumor types. The Y-axis represents the number of positive cells per 1.15 mm² spot, and the results are shown as means plus standard error.

Example 30 Immunohistochemical Analysis of HLA-E Expression in 7 Tumor Types

To evaluate HLA-E expression, a commercial anti-HLA-E antibody (clone MEM-E/02) was validated for immunohistochemistry in formalin-fixed paraffin-embedded (FFPE) tissues. HLA-E was broadly expressed in tumor samples examined. Positive staining was observed in both tumor epithelium and stroma. Whole slide image analysis using HALO™ software revealed a relatively higher abundance of HLA-E in HNSCC and NSCLC, and a lower abundance in pancreatic carcinoma and colorectal cancer (CRC), as shown in FIG. 47. FIG. 47 shows the results of whole slide image analysis of HLA-E expression using HALO software in 13 HPV+ head and neck squamous cell carcinoma (NNSCC/HPV+), 11 HPV− head and neck squamous cell carcinoma (HNSCC/HPV−), 10 non-small cell lung adenocarcinoma (NSCLC-ADC), 10 HNSCC, 10 non-small cell lung squamous cell carcinoma (NSCLC-SCC), 6 pancreatic carcinoma, and 10 colon carcinoma (CRC) samples. Immunoreactivity of HLA-E was calculated as follows: total area of ‘brown’ x average OD of ‘brown’/total area of tissue. Data was normalized to mm². The results are shown as means plus standard error.

In tumor epithelial cells, the positive staining was primarily cytoplasmic. Notable membrane staining was also exhibited in some tumor cells. In tumor stroma, positive staining was primarily distributed in mononuclear cells and subsets of interstitial cells, as well as occasionally in microvasculature and neutrophils in some cases. In general, HLA-E expression in stroma was present in almost all cases. However, the expression in tumor epithelial cells varied significantly from sample to sample in all tumor types examined. High-level tumor epithelial expression was seen in most cases, while negative or very low expression was seen in small number of cases, as shown in FIG. 48. In FIG. 48, “St” are tumor stroma, and “TuEp” are tumor epithelial cells. FIG. 48 shows representative images of HLA-E expression in multiple tumors. Immunohistochemistry with a validated commercial monoclonal antibody on FFPE sections from HPV+ head and neck squamous cell carcinoma (NNSCC/HPV+), non-small cell lung adenocarcinoma (NSCLC-ADC), non-small cell squamous cell carcinoma (NSCLC-SCC), and colon carcinoma (CRC).

Patients with Virus-Derived Tumors:

Viral infections can induce NKG2A expression on NK cells, as was observed in hepatitis C chronically infected patients. (Harrison R J et al., “Association of NKG2A with treatment for chronic hepatitis C virus infection.” Clin Exp Immuno 161:306-14 (2010)). In addition, the frequency of NKG2A+ CD8 T cells was shown to be higher in TIL from patients with HPV-16+ head and neck carcinomas, compared to HPV-16 tumors. (van Hall T, et al. “NKG2A checkpoint receptor expression on tumor-infiltrating CD8+ T cells restrains efficacy of immunotherapy.” Cancer Res Supplement; Abstract 2999 (2017)). We evaluated an association between HPV status in head and neck tumors and expression of the NKG2A/HLA-E pathway by measuring HLA-E expression by IHC between biopsies from HPV+ and HPV− head and neck tumors. Our initial assessment in a relatively small number of samples showed that HLA-E expression by IHC was not significantly different between HPV+ and HPV− samples, as shown in FIG. 48. We will test HPV status in head and neck tumors and expression of the NKG2A/HLA-E pathway by measuring CD8 (as a surrogate for NKG2A expression) and HLA-E expression by IHC between biopsies from HPV+ and HPV− head and neck tumors. We also continued the IHC analysis with additional tumor types and larger sample sizes, and this analysis is described in Example 31 below.

Example 31 Immunohistochemical Analysis of HLA-E Expression in 17 Tumor Types

We profiled HLA-E expression across 17 tumor types by immunohistochemical (IHC) assessment. We believe that cancer patients are most likely to respond to anti-tumor treatment with the anti-NKG2A antibodies described herein for tumor types that express higher levels of HLA-E.

To assess HLA-E expression, immunohistochemistry (IHC) was performed on formalin-fixed paraffin-embedded (FFPE) sections from 17 tumor types/subtypes, including cervical, bladder, breast, colorectal adenocarcinoma (CRC), endometrial, head & neck squamous cell carcinoma (HNSCC), gastric, glioblastoma (GBM), melanoma, non-Hodgkin lymphoma (follicular lymphoma), ovarian, renal cell (RCC), pancreatic, prostate, small cell lung carcinoma (SCLC), non-small cell lung adenocarcinoma (NSCLC-AD), and non-small cell lung squamous cell carcinoma (NSCLC-SQC), with well-characterized commercial monoclonal antibody (clone MEM/02). There were 14 to 43 samples per tumor type/subtype. Stained slides were evaluated by the study pathologists to identify the tissue or cell type. All slides were judged for adequacy of tissue elements and staining. A conventional/manual scoring was performed by study pathologists.

The results showed that HLA-E expression was observed in the tumor cell, immune, and endothelial cell compartments across various indications. In tumor cells, positive staining was predominantly cytoplasmic across human tumor types with limited expression noted at the surface plasma membrane. Total HLA-E expression ranged from diffuse tumor cell positivity to heterogeneous expression to some cases with low to almost no HLA-E expression. FIG. 57 shows a boxplot of total HLA-E positivity score across 17 different tumor types as evaluated by immunohistochemistry. Total HLA-E score is defined as percent of combined cytoplasmic and/or membrane HLA-E positivity in the tumor cells. The data used to generate FIG. 57 is shown in the Table below.

Total Total Tumor Tumor HLA-E HLA-E Tumor Samples Positive Positive Standard 25^(th) 75^(th) Type (N) Score¹ Percentage² Mean Median Deviation Percentile Percentile Bladder 20 17 85% 37.3 40.0 30.4 11.5 52.5 Cancer Breast 20 14 70% 16.3 7.5 22.9 0 21.25 Cancer Glioblastoma 19 16 84.21%   9.9 5.0 15.1 2 10 Multiforme Gastric 20 17 85% 32.7 15.0 36.4 2.75 58.75 Cancer Kidney 20 15 75% 20.4 9.0 23.4 1.5 37.5 Cancer, Clear Cell Renal Cell NSCLC-AD 40 38 95% 35.8 25.0 33.6 6 61.25 NSCLC-SCC 40 37 92.50%   39.1 30.0 32.0 10 65 Melanoma 20 18 90% 32.0 10.0 35.0 1.88 62.5 Endometrial 20 19 95% 27.3 12.5 31.9 3 41.25 Cancer Ovarian 20 20 100%  25.0 16.5 25.0 9.88 25 Cancer CRC 39 38 97.44%   30.2 20.0 28.7 8 47.5 NHL, 20 20 100%  58.3 61.3 26.3 32.38 77.5 Follicular Lymphoma HNSCC 40 39 97.50%   48.7 46.5 30.9 19 76.25 Prostate 20 20 100%  5.4 4.5 4.4 2.38 5.5 Cancer Cervical 20 20 100%  54.7 62.5 27.2 28.75 75 Cancer Pancreatic 43 41 95.35%   44.1 42.5 33.7 10 72.5 SCLC 14 9 64.29%   14.9 4.5 20.6 0 23.75 ¹To calculate the “Total Tumor HLA-E Positive score,” a 1% cutoff on the HLA-E values was used. In other words, if greater than or equal to 1% of the tumors were HLA-E positive (cytoplasmic and/or membrane HLA-E), then the sample was labeled as “positive.” ²“Total tumor HLA-E positive percentage” is the percentage of HLA-E positive patients in the tumor type. In FIG. 57, each of the boxes in the boxplots show the total tumor HLA-E positive value at the 25^(th) percentile; the middle line of each of the boxes is the median value; and the upper limit of each of the boxes show the value at the 75^(th) percentile.

In conclusion, there was a dynamic range of total HLA-E expression across and within the tumor indications. We believe that cancer patients with high HLA-E expression in tumor cells are more likely to respond to anti-tumor treatment with the anti-NKG2A antibodies described herein. Non-Hodgkin lymphoma (follicular lymphoma), cervical, HNSCC, pancreatic, NSCLC, and bladder tumors had the highest level of total HLA-E expression; thus, patients with these cancers have the highest likelihood of responding to anti-tumor treatment with the anti-NKG2A antibodies described herein. This study provided tumor profiling data to support the prioritization of indication selection to include, for example, HNSCC, RCC, NSCLC, and CRC cancers in clinical development.

Prostate, GBM, and SCLC demonstrated the lowest level of HLA-E expression. Nevertheless, we believe that patients with tumor types that have lower HLA-E expression will also respond to anti-tumor treatment with the anti-NKG2A antibodies described herein. This study is described in more detail below.

Materials and Methods Tissue Samples

Overall, 17 cancer types/subtypes from two sets of samples were studied. Full size FFPE slides of 6 tumor types/subtypes, including colorectal adenocarcinoma (CRC), head and neck squamous cell carcinoma (HNSCC), non-small cell lung adenocarcinoma (NSCLC-AD), non-small cell lung squamous cell carcinoma (NSCLC-SQC), pancreatic carcinoma, and small cell lung carcinoma (SCLC), with 14-24 samples per tumor type, were studied. FFPE tissue samples were obtained from various commercial tissue vendors (BioIVT, Detroit, Mich.; BioChain Institute Inc. Newark, Calif.; Conversant Biologics Inc, Huntsville, Ala.; Cooperative Human Tissue Network, Philadelphia, Pa.; Indivumed GmbH, Lewisburg, Pa.; The MT Group Inc. Van Nuys, Calif.; TriStar Technology Group LLC, Rockville, Md.). In addition, multi-tumor blocks (MTBs) containing 16 tumor types/subtypes including bladder, breast, GBM, gastric, RCC, NSCLC-AD, NSCLC-SQC, melanoma, endometrial, ovarian, CRC, HNSCC, prostate, cervical, pancreatic carcinoma, and non-Hodgkin lymphoma (follicular lymphoma), with 20 cases/tumor types, were studied. Each MTB contained 5 cases of a single indication per FFPE block and 1 hyperplastic tonsil as positive control. The MTBs were made by Folio Biosciences (now Discovery Life Sciences) for BMS.

Antibodies and Reagents

-   -   Mouse monoclonal antibody anti-human HLA-E, Clone MEM/02 (Abcam,         Cambridge, Mass., Catalog ab2216, Lot GR251472-15)     -   Mouse IgG1 Isotype, Clone 11711 (R&D Systems, Minneapolis,         Minn., Catalog MAB002, Lot IX2415091)     -   Bond Polymer Refine Detection (Leica Biosystems, Buffalo Grove,         Ill., Catalog DS9800, Lot 46396 and 46794)     -   Bond Epitope Retrieval 2 (Leica Biosystems, Buffalo Grove, Ill.,         Catalog AR9640, Lot ER20180)     -   Protein Block, Serum-Free (Agilent Technologies, Santa Clara,         Calif., Catalog X0909, Lot 10117172) Human gamma globulin         (Sigma-Aldrich, St. Louis, Mo., Catalog G-4386, Lot SLV M0524V)

IHC Methods

FFPE sections at 4 μm were prepared, paraffin dipped and stored at 4° C. until use. For MTBs, 4 μm sections were cut and stored at 4° C. in vacuum sealed bags until use. All IHC assays including respective isotype controls were optimized for automated IHC using the Leica BondRX platform. FFPE sections from the same hyperplastic human tonsil tissue block were used as positive controls for all IHC runs.

For immunostaining, FFPE slides were baked, deparaffinized in xylenes, and rehydrated through a graded ethanol series following routine histology procedures. Antigen retrieval was performed on the Leica Bond RX instrument. Automated IHC staining procedures were carried out at room temperature on the Leica Bond RX. Briefly, slides were blocked in peroxidase block for 10 minutes followed by non-specific protein block for 20 minutes. Primary antibodies were incubated for 60 minutes followed by Refine Linker for 30 minutes. Slides were then incubated with Refine Polymer for 30 minutes. Finally, slides were reacted with the DAB substrate-chromogen solution for 6 minutes, counterstained for 8 minutes with Leica Hematoxylin, and then dehydrated, cleared, and coverslipped with Permount following routine histological procedure.

After immunostaining, all slides were scanned at 20× using the Aperio AT2 whole slide scanner (Leica Biosystems Imaging, Vista, Calif.), and the images were stored and organized with the Aperio eSlide Manager Software.

Pathology Assessment and Manual Scoring Methods

The study pathologist evaluated the slides to determine tissue adequacy and staining quality. The study pathologist manually scored the HLA-E IHC stained slides and reviewed isotype and positive control slides. Pathology assessment consisted of H&E evaluation to determine the tumor content and confirm tumor type. HLA-E immunohistochemistry (IHC) stained slides were scored manually using whole slide scanned images for tumor and immune cell positivity. No background staining was observed in the isotype control slides. HLA-E expression in tumor cells (TC), immune cells (IC) and endothelial cells were evaluated. Percentage of cytoplasmic HLA-E and membrane HLA-E were recorded. The total HLA-E score was the key reportable data for HLA-E expression, which is composed of combined cytoplasmic and/or membrane HLA-E positivity in the tumor cells, and was reported as percent HLA-E positive of total tumor cells. The percent HLA-E immune cell positivity along with predominant geographical location of HLA-E positive immune cells and endothelium were also assessed.

Results

HLA-E was diffusely positive in hyperplastic tonsil. HLA-E membranous/cytoplasmic staining was noted within the crypt epithelium, germinal centers, and interfollicular zones with strongest intensity in the mantle zones. In the tumor tissues, specific HLA-E expression was observed in tumor cells, immune cells, and endothelial cells. Cytoplasmic and membranous localization of HLA-E were noted within the tumor cells across indications and exhibited a dynamic range of intensity ranging from weak (1+ intensity) to strong (3+ intensity). HLA-E expression was predominantly cytoplasmic across human tumor types with limited expression noted at the plasma membrane. Total HLA-E expression ranged from diffuse tumor cell positivity to heterogeneous expression to some cases with low to very low HLA-E expression. Cervical, HNSCC, pancreatic, NSCLC, bladder, and non-Hodgkin lymphoma tumors had the highest level of total HLA-E expression with prostate, GBM and SCLC being the lowest, as shown in FIG. 59. RCC had the highest level of expression of membranous HLA-E among tumor types examined, although the total HLA-E level was relatively low.

Conclusion

In conclusion, the data showed a dynamic range of expression of total HLA-E across and within various tumor indications. Cervical, HNSCC, pancreatic, NSCLC, and bladder tumors had the highest level of total HLA-E expression, while prostate, GBM and SCLC demonstrated the lowest frequency of expression. This study provided tumor profiling data to support the prioritization of indication selection to include, for example, HNSCC, RCC, NSCLC, and CRC cancers in the clinical development of anti-NKG2A antibodies described herein. In some embodiments, we believe that patients with tumor types that have lower HLA-E expression will also respond to anti-tumor treatment with the anti-NKG2A antibodies described herein.

Example 32

Patients with High Serum Levels of Soluble HLA-E

Cancer patients with melanoma show greater titers of soluble HLA-E in their serum compared to healthy donors. (Allard M et al., “Serum soluble HLA-E in melanoma: a new potential immune-related marker in cancer,” PLoS One, 6:e21118 (2011)).

Recent data from a nivolumab clinical trial in patients with NSCLC (Checkmate 063) suggest that patients with high sHLA-E have a lower response rate to nivolumab. (Rebmann V et al., “Soluble HLA-G and -E (sHLA-G/E) as potential biomarkers of clinical outcomes in patients (pts) with advanced, refractory squamous (SQ) NSCLC treated with nivolumab (NIVO): CheckMate 063”, Cancer Res; AM2017-CT126 (2017)).

We believe that patients with a high level of HLA-E expression in tumors are more likely to benefit from treatment with the anti-NKG2A antibodies described herein, including the NKG2A.9 antibody, either alone or in combination with, for example, nivolumab. In some embodiments, a healthcare provider selects patients with a specific level of HLA-E, such as soluble HLA-E, for treatment with the anti-NKG2A.9 antibodies described herein. In other embodiments, a healthcare provider selects patients with a specific level of CD8+ expression (as a surrogate for NKG2A expression) for treatment with the anti-NKG2A.9 antibodies described herein.

A selective and sensitive SIMOA-based immunoassay to measure sHLA-E (LLOQ: 11 pg/mL) was developed and used to test two independent sample sets: (A) a first set that contains sera and plasma from healthy controls (n=17) and from patients from six cancer types (n=10 patients per cancer type) (FIG. 49A) and (B) a second set of 100 healthy controls and 100 lung cancer patients (FIG. 49B). In both sets, interpatient variability was observed, with similar ranges in healthy donors and cancer patients. This assay will confirm soluble HLA-E levels as a predictive marker of response to nivolumab using this assay.

Within each tumor type, significant inter-patient variability for HLA-E expression was observed, as shown in FIG. 49. We believe patients with higher tumor HLA-E and higher CD8+ (using CD8+ as a surrogate for NKG2A expression) will benefit from NKG2A inhibition to restore antitumor activity of CD8+ T and NK cells.

Example 33

Phase 1/Phase 2 Study with the NKG2A.9 Antibody

We will conduct a Phase I/II study with the NKG2A.9 antibody to develop a safe and efficacious therapy to treat cancer. Although not bound by any mechanism, the therapy will restore T cell response to checkpoint inhibitors in PD-L1 resistant/refractory tumors and/or enhance NK activity in NK-enriched solid tumors.

The clinical development plan is summarized in Table 9 below.

TABLE 9 Anti-NKG2A Antibody Clinical Development Plan Anti-NKG2A Antibody with Nivolumab Checkpoint blockade with anti-NKG2A Selection of patients with: antibody in combination with nivolumab Inflamed or high-TMB tumors with known will synergize to extend the benefit and anti-PD-1 activity but less than complete deepen response in tumors resistant or response AND high expression of HLA-E refractory to anti-PD-L1 therapy or and/or CD8 (as a surrogate for NKG2A associated with poor response to anti-PD-L1 expression); and/or therapy Tumors associated with a viral etiology AND high expression of CD8 (NKG2A surrogate) and/or HLA-E; and/or Tumors that relapse after anti-PD-1 treatment AND have high expression of CD8 (NKG2A surrogate) and/or total tumor HLA-E. Anti-NKG2A Antibody with Other I-O Agents and Multi-Checkpoint Combinations Anti-NKG2A antibody in combination with Selection of patients with: multiple checkpoint inhibitors will Inflamed or high-TMB tumors with expression overcome I-O resistance by redundant of multiple immune checkpoint proteins checkpoint pathways and optimize clinical benefit. The clinical development of the NKG2A.9 antibody will begin with a first in human (FIH), Phase ½ study. An initial monotherapy dose escalation will be performed to determine the safety, maximum tolerated dose (MTD) or maximum administered dose, PK, PD, and preliminary evaluation of clinical activity. Intravenous administration route will initially be used in the study, with subcutaneous administration in other arms of the study. Alternatively, an intravenous administration route can be used. With a staggered start, a parallel dose escalation will occur with the NKG2A.9 antibody combination with nivolumab, as illustrated in FIG. 50. The safety, MTD or maximum administered dose, PK, PD, and preliminary evaluation of clinical activity will next be evaluated with the combination therapy. Triplet checkpoint blockade combinations will also be evaluated during dose escalation and include the NKG2A.9 antibody, nivolumab, and other checkpoint inhibitors including but not limited to relatlimab (anti-LAG-3), anti-TIM-3, anti-TIGIT, ipilimumab, CTLA-4 NF or Probody, or other anti-cancer therapies, including, for example, chemotherapy, signal transduction agents such as cetuximab, amongst other therapies.

Patient/tumor selection for escalation and expansion cohorts will be based on ongoing analysis of The Cancer Genome Atlas (TCGA) expression profiles, expression profiles from internal nivolumab trials, profiling of tumor samples for CD8 levels (as a surrogate for NKG2A levels), and profiling of tumor and serum samples for HLA-E, including virally-induced tumors. For example, patient selection will be based on choosing patients whose tumors are CD8+ (as a surrogate for NKG2A) and HLA-E+, as illustrated in FIG. 50. In another embodiment, patient selection will also be based on tumors with high inflammation or tumor mutation burden.

Patients enrolled in this study will be required to provide pre-treatment and on-treatment biopsies as well as serial peripheral blood samples to test the hypothesis that treatment with anti-NKG2A alone or in combination with nivolumab increases the number or functional activities of CD8+ T and NK cells in the tumor. Particular attention will be dedicated to analyzing whether such changes in the tumor immune microenvironment correlate with clinical response. Pharmacodynamic studies will evaluate markers of target engagement, T cell activation, changes in NKG2A surface expression, IFN-γ, and serum sHLA-E. Subjects with NKG2A-positive and HLA-E+ tumors (including but not limited to non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), and head and neck squamous cell carcinoma (HNSCC)) and tumors of viral origin (head and neck, cervical, hepatocellular cancers) will optionally be included. Subjects may have tumors that are resistant/refractory to anti-PD-1/PD-L1 therapy or tumors that are reported to be insensitive to anti-PD-1/PD-L1 therapy. Alternatively, subjects may be naïve to PD-1/PD-L1 treatment.

The Sequence Listing is provided in the Table below.

Sequence Listing SEQ ID NO Name Sequence   1 Human ATGGATAACC AAGGAGTAAT CTACTCAGAC CTGAATCTGC CCCCAAACCC 50 NKG2A AAAGAGGCAG CAACGAAAAC CTAAAGGCAA TAAAAACTCC ATTTTAGCAA 100 nucleotide CTGAACAGGA AATAACCTAT GCGGAATTAA ACCTTCAAAA AGCTTCTCAG 150 sequence GATTTTCAAG GGAATGACAA AACCTATCAC TGCAAAGATT TACCATCAGC 200 (CCDS8625.1) TCCAGAGAAG CTCATTGTTG GGATCCTGGG AATTATCTGT CTTATCTTAA 250 TGGCCTCTGT GGTAACGATA GTTGTTATTC CCTCTACATT AATACAGAGG 300 CACAACAATT CTTCCCTGAA TACAAGAACT CAGAAAGCAC GTCATTGTGG 350 CCATTGTCCT GAGGAGTGGA TTACATATTC CAACAGTTGT TACTACATTG 400 GTAAGGAAAG AAGAACTTGG GAAGAGAGTT TGCTGGCCTG TACTTCGAAG 450 AACTCCAGTC TGCTTTCTAT AGATAATGAA GAAGAAATGA AATTTCTGTC 500 CATCATTTCA CCATCCTCAT GGATTGGTGT GTTTCGTAAC AGCAGTCATC 550 ATCCATGGGT GACAATGAAT GGTTTGGCTT TCAAACATGA GATAAAAGAC 600 TCAGATAATG CTGAACTTAA CTGTGCAGTG CTACAAGTAA ATCGACTTAA 650 ATCAGCCCAG TGTGGATCTT CAATAATATA TCATTGTAAG CATAAGCTTT 700 AG 702   2 Human MDNQGVIYSD LNLPPNPKR QQRKPKGNKNS ILATEQEITY AELNLQKASQ 50 NKG2A DFQGNDKTYH CKDLPSAPE KLIVGILGIIC LILMASVVTI VVIPSTLIQR 100 amino acid HNNSSLNTRT QKARHCGHC PEEWITYSNSC YYIGKERRTW EESLLACTSK 150 sequence NSSLLSIDNE EEMKFLSII SPSSWIGVFRN SSHHPWVTMN GLAFKHEIKD 200 (GenBank SDNAELNCAV LQVNRLKSA QCGSSIIYHCK HKL 233 NP_002250.2   3 Human MSKQRGTFSE VSLAQDPKRQ QRKPKGNKSS ISGTEQEIFQ VELNLQNPSL 50 NKG2C NHQGIDKIYD CQGLLPPPEK LTAEVLGIIC IVLMATVLKT IVLIPFLEQN 100 amino acid NSSPNTRTQK ARHCGHCPEE WITYSNSCYY IGKERRTWEE SLLACTSKNS 150 sequence SLLSIDNEEE MKFLASILPS SWIGVFRNSS HHPWVTINGL AFKHKIKDSD 200 (GenBank NAELNCAVLQ VNRLKSAQCG SSMIYHCKHK L 231 CAA04922.1) NKG2A.9 Antibody (13F3.A4 antibody with VH-I107T framework reversion and VK-N30S to remove deamidation site) (SEQ ID NOs: 4-17, 163)   4 NKG2A.9 gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga 50 light chain cagagtcacc atcacttgcc gggcaagtca gggcatttcc agtgctttag 100 nucleotide cctggtatca gcagaaacca gggaaagctc ctaagctcct gatctatgat 150 sequence gcctccagtt tgaaaagtgg ggtcccatca aggttcagcg gcagtggatc 200 (including tgggacagat ttcactctca ccatcagcag cctgcagcct gaagattttg 250 C-terminal caacttatta ctgtcaacag tttaatagtt accctctcac cttcggccaa 300 ″tag″ stop gggacacgac tggagattaa acgtacggtg gctgcaccat ctgtcttcat 350 codon) cttcccgcca tctgatgagc agttgaaatc tggaactgcc tctgttgtgt 400 gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg 450 gataacgccc tccaatcggg taactcccag gagagtgtca cagagcagga 500 cagcaaggac agcacctaca gcctcagcag caccctgacg ctgagcaaag 550 cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645   5 NKG2A.9 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 light chain ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 amino acid GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 sequence DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 LSSPVTKSFN RGEC 214   6 NKG2A.9 gaggtgcagc tggtggagtc tgggggaggc ctggtcaagc cgggggggtc 50 heavy chain cctgagactc tcctgtgcag cctctggatt caccttcagt tcccatagta 100 nucleotide tgaactgggt ccgccaggct ccagggaagg ggctggagtg ggtctcagcc 150 sequence ataagtagta gtagtagtta catatactac gcagactcag tgaagggccg 200 (same as attcaccatc tccagagaca acgccaagaa ctcactgtat ctgcaaatga 250 NKG2A.11- acagcctgag agccgaggac acggctgtgt attactgtgc gagagaagag 300 G1.3f heavy tgggggctac cctttgacta ctggggccag ggaaccctgg tcaccgtctc 350 chain ctcagctagc accaagggcc catcggtctt ccccctggca ccctcctcca 400 nucleotide agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac 450 sequence) ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg 500 (including cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctca 550 C-terminal gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 ″tga″ stop tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agagagttga 650 codon) gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgcc cagcacctg 700 aagccgaagg ggccccgtca gtcttcctct tccccccaaa acccaaggac 750 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt 800 gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg 850 aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg 950 caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg 1000 agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1050 accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac 1100 ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga 1150 gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag 1250 gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 1300 acaaccacta cacgcagaag agcctctccc tgtccccggg ttga 1344   7 NKG2A.9 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 heavy chain ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 amino acid WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 sequence FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 (same as CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 NKG2A.11- TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 G1.3f heavy YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 chain amino TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 acid SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPG 447 sequence) (terminal lysine is absent) 163 NKG2A.9 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 heavy chain ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 amino acid WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 sequence FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 (same as CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 NGK2A11- TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 G1.3f heavy YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 chain amino TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 acid SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 447 sequence) (shown with the terminal lysine)   8 NKG2A.9 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 heavy chain ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 variable WGLPFDYWGQ GTLVTVSS 118 domain (same as NKG2A.11 heavy chain variable domain)   9 NKG2A.9 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 light chain ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 variable GTRLEIK 107 domain  10 NKG2A.9 SHSMN 5 VH CDR1  11 NKG2A.9 AISSSSSYIY YADSVKG 17 VH CDR2  12 NKG2A.9 EEWGLPFDY 9 VH CDR3  13 NKG2A.9 RASQGISSAL A 11 VL CDR1  14 NKG2A.9 DASSLKS 7 VL CDR2  15 NKG2A.9 QQFNSYPLT 9 VL CDR3  16 NKG2A.9 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 50   heavy chain HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP 100 constant KSCDKTHTCP PCPAPEAEGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 150 domain HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 200 EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC 250 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEALHNHYT QKSLSLSPG 329  17 NKG2A.9 RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 light chain NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 constant SFNRGEC 107 domain NKG2A.11 Antibody (13F3.A4 VH-I107T, VK-N30P) (13F3.A4 with VH-I107T framework  reversion and VK-N30P to remove deamidation site) (SEQ ID NOs: 18-21, 164-165) (SEQ ID NOs: 6-7 (NKG2A.11 heavy chain sequences nucleotide and amino acid  sequences, respectively))  18 NKG2A.11 gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga 50 light chain cagagtcacc atcacttgcc gggcaagtca gggcattccc agtgctttag 100 nucleotide cctggtatca gcagaaacca gggaaagctc ctaagctcct gatctatgat 150 sequence gcctccagtt tgaaaagtgg ggtcccatca aggttcagcg gcagtggatc 200 (including tgggacagat ttcactctca ccatcagcag cctgcagcct gaagattttg 250 C-terminal caacttatta ctgtcaacag tttaatagtt accctctcac cttcggccaa 300 ″tag″ stop gggacacgac tggagattaa acgtacggtg gctgcaccat ctgtcttcat 350 codon) cttcccgcca tctgatgagc agttgaaatc tggaactgcc tctgttgtgt 400 gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg 450 gataacgccc tccaatcggg taactcccag gagagtgtca cagagcagga 500 cagcaaggac agcacctaca gcctcagcag caccctgacg ctgagcaaag 550 cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgcccgtcacaaa gagcttcaac aggggagagt gttag 645  19 NKG2A.11 AIQLTQSPSS LSASVGDRVT ITCRASQGIP SALAWYQQKP GKAPKLLIYD 50 light chain ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 amino acid GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 sequence DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 LSSPVTKSFN RGEC 214 164 NKG2A.11 AIQLTQSPSS LSASVGDRVT ITCRASQGIP SALAWYQQKP GKAPKLLIYD 50 light chain ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 variable GTRLEIK 107 domain amino acid sequence 165 NKG2A.11 RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 light chain NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 constant SFNRGEC 107 domain amino acid sequence  20 Osteonectin atgagggctt ggatcttctt tctgctctgc ctggccgggc gcgccttggc 50 signal c 51 nucleotide sequence  21 Osteonectin MRAWIFFLLC LAGRALA 17 signal amino acid sequence NKG2A.6 Antibody (13F3.A4 antibody) (SEQ ID NOs: 22-36, 166-169)  22 Signal MELGLRWVFL VAILEGVQC 19 amino acid sequence for 13F3.A4 heavy chain  23 13F3.A4 ATGGAACTGG GGCTCCGCTG GGTTTTCCTT GTTGCTATTT TAGAAGGTGT -57 VH(heavy CCAGTGT -7 chain GAGGTGCAGC TGGTGGAGTC TGGGGGAGGC CTGGTCAAGC CGGGGGGGTC 50 variable CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT TCCCATAGTA 100 region TGAACTGGGT CCGCCAGGCT CCAGGGAAGG GGCTGGAGTG GGTCTCAGCC 150 nucleotide ATAAGTAGTA GTAGTAGTTA CATATACTAC GCAGACTCAG TGAAGGGCCG 200 sequence) ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTAT CTGCAAATGA 250 (with 57- ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAAGAG 300 nucleotide TGGGGGCTAC CCTTTGACTA CTGGGGCCAG GGAATCCTGG TCACCGTCTC 350 signal CTCA 354 sequence) 166 13F3.A4 GAGGTGCAGC TGGTGGAGTC TGGGGGAGGC CTGGTCAAGC CGGGGGGGTC 50 VH (heavy CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT TCCCATAGTA 100 chain TGAACTGGGT CCGCCAGGCT CCAGGGAAGG GGCTGGAGTG GGTCTCAGCC 150 variable ATAAGTAGTA GTAGTAGTTA CATATACTAC GCAGACTCAG TGAAGGGCCG 200 region ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTAT CTGCAAATGA 250 nucleotide ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAAGAG 300 sequence) TGGGGGCTAC CCTTTGACTA CTGGGGCCAG GGAATCCTGG TCACCGTCTC 350 (without CTCA 354 signal sequence)  24 13F3.A4 MELGLRWVFL VAILEGVQC -19 VH heavy EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 chain ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 variable WGLPFDYWGQ GILVTVSS 118 region amino acid sequence (with 19- amino acid signal sequence) 167 13F3.A4 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 VH amino ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 acid WGLPFDYWGQ GILVTVSS 118 sequence (without signal sequence)  25 13F3.A4 VL ATGGACATGA GGGTCCCCGC TCAGCTCCTG GGGCTTCTGC TGCTCTGGCT -66 nucleotide CCCAGGTGCC AGATGT -16 sequence GCCATCCAGT TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA 50 (with 66- CAGAGTCACC ATCACTTGCC GGGCAAGTCA GGGCATTAAC AGTGCTTTAG 100 nucleotide CCTGGTATCA GCAGAAACCA GGGAAAGCTC CTAAGCTCCT GATCTATGAT 150 signal GCCTCCAGTT TGAAAAGTGG GGTCCCATCA AGGTTCAGCG GCAGTGGATC 200 sequence) TGGGACAGAT TTCACTCTCA CCATCAGCAG CCTGCAGCCT GAAGATTTTG 250 CAACTTATTA CTGTCAACAG TTTAATAGTT ACCCTCTCAC CTTCGGCCAA 300 GGGACACGAC TGGAGATTAA A 321 168 13F3.A4 VL GCCATCCAGT TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA 50 nucleotide CAGAGTCACC ATCACTTGCC GGGCAAGTCA GGGCATTAAC AGTGCTTTAG 100 sequence CCTGGTATCA GCAGAAACCA GGGAAAGCTC CTAAGCTCCT GATCTATGAT 150 (without GCCTCCAGTT TGAAAAGTGG GGTCCCATCA AGGTTCAGCG GCAGTGGATC 200 signal TGGGACAGAT TTCACTCTCA CCATCAGCAG CCTGCAGCCT GAAGATTTTG 250 sequence) CAACTTATTA CTGTCAACAG TTTAATAGTT ACCCTCTCAC CTTCGGCCAA 300 GGGACACGAC TGGAGATTAA A 321  26 13F3.A4 VL MDMRVPAQLL GLLLLWLPGA RC -22 (light chain AIQLTQSPSS LSASVGDRVT ITCRASQGIN SALAWYQQKP GKAPKLLIYD 50 variable ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 region GTRLEIK 107 amino acid sequence) (with 22- amino acid signal sequence) 169 13F3.A4VL AIQLTQSPSS LSASVGDRVT ITCRASQGIN SALAWYQQKP GKAPKLLIYD 50 amino acid ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 sequence GTRLEIK 107 (without signal sequence)  27 13F3.A4 SHSMN 5 VH CDR1  28 13F3.A4 AISSSSSYIY YADSVKG 17 VH CDR2  29 13F3.A4 EEWGLPFDY 9 VH CDR3  30 13F3.A4 VL RASQGINSAL A 11 CDR1  31 13F3.A4 VL DASSLKS 7 CDR2  32 13F3.A4 VL QQFNSYPLT 9 CDR3  33 13F3.A4 CH ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 50 heavy chain HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP 100 constant KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 150 region HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 200 EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 250 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 330  34 13F3.A4 CL RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 light chain NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 constant SFNRGEC 107 region  35 13F3.A4 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 heavy chain ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 amino acid WGLPFDYWGQ GILVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 sequence FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 (without CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD 250 signal TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 sequence) YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 Terminal TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 lysine can SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPG(K) 448 be absent.  36 13F3.A4 AIQLTQSPSS LSASVGDRVT ITCRASQGIN SALAWYQQKP GKAPKLLIYD 50 light chain ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 amino acid GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 sequence DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 (without LSSPVTKSFN RGEC 214 signal sequence)  37 11H9.A1 ATGGAACTGG GGCTCCGCTG GGTTTTCCTT GTTGCTATTT TAGAAGGTGT -57 VH CCAGTGT -7 (heavy chain GAGGTGCAGT TGGTGGAGTC TGGGGGAGGC CTGGTCAAGC CTGGGGGGTC 50 variable CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT AGCTATAGCA 100 region TGAACTGGGT CCGCCAGGCT CCAGGGAAGG GGCTGGAGTG GGTCTCATCC 150 nucleotide ATTAGTAGTA GTAGTAGTTA CATATACTAC GCAGACTCAG TGAAGGGCCG 200 sequence) ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTAT CTGCAAATGA 250 (with 57- ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGACTACTA 300 nucleotide TGGTTCGGGG AGATTTTTGA CTACTGGGGC CAGGGAACCC TGGTCACCGT 350 signal CTCCTCA 357 sequence) 170 11H9.A1 GAGGTGCAGT TGGTGGAGTC TGGGGGAGGC CTGGTCAAGC CTGGGGGGTC 50 VH CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT AGCTATAGCA 100 (heavy chain TGAACTGGGT CCGCCAGGCT CCAGGGAAGG GGCTGGAGTG GGTCTCATCC 150 variable ATTAGTAGTA GTAGTAGTTA CATATACTAC GCAGACTCAG TGAAGGGCCG 200 region ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTAT CTGCAAATGA 250 nucleotide ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGACTACTA 300 sequence) TGGTTCGGGG AGATTTTTGA CTACTGGGGC CAGGGAACCC TGGTCACCGT 350 (without CTCCTCA 357 signal sequence)  38 11H9.A1 MELGLRWVFL VAILEGVQC -19 VH (heavy EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS 50 chain ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARLL 100 variable WFGEIFDYWG QGTLVTVSS 119 region amino acid sequence) (with 19- amino acid signal sequence) 171 11H9.A1 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS 50 VH (heavy ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARLL 100 chain WFGEIFDYWG QGTLVTVSS 119 variable region amino acid sequence) (without signal sequence)  39 11H9.A1 ATGGACATGA GGGTCCCCGC TCAGCTCCTG GGGCTTCTGC TGCTCTGGCT -66 VL (light CCCAGGTGCC AGATGT -16 chain GCCATCCAGT TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA 50 variable CAGAGTCACC ATCACTTGCC GGGCAAGTCA GGGCATTAGC AGTGCTTTAG 100 region CCTGGTATCA GCAGAAACCA GGGAAAGCTC CTAAGCTCCT GATCTATGAT 150 nucleotide GCCTCCAGTT TGAAAAGTGG GGTCCCATCA AGGTTCAGCG GCAGTGGATC 200 sequence) TGGGACAGAT TTCACTCTCA CCATCAGCAG CCTGCAGCCT GAAGATTTTG 250 (with 66- CAACTTATTA CTGTCAACAG TTTAATAGTT ACCCGATCAC CTTCGGCCAA 300 nucleotide GGGACACGAC TGGAGATTAA A 321 signal sequence) 172 11H9.A1 GCCATCCAGT TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA 50 VL (light CAGAGTCACC ATCACTTGCC GGGCAAGTCA GGGCATTAGC AGTGCTTTAG 100 chain CCTGGTATCA GCAGAAACCA GGGAAAGCTC CTAAGCTCCT GATCTATGAT 150 variable GCCTCCAGTT TGAAAAGTGG GGTCCCATCA AGGTTCAGCG GCAGTGGATC 200 region TGGGACAGAT TTCACTCTCA CCATCAGCAG CCTGCAGCCT GAAGATTTTG 250 nucleotide CAACTTATTA CTGTCAACAG TTTAATAGTT ACCCGATCAC CTTCGGCCAA 300 sequence) GGGACACGAC TGGAGATTAA A 321 (without signal sequence)  40 11H9.A1 MDMRVPAQLL GLLLLWLPGA RC -22 VL AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 (light chain ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPITFGQ 100 variable GTRLEIK 107 region amino acid sequence) (with 22- amino acid signal sequence) 173 11H9.A1 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 VL ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPITFGQ 100 (light chain GTRLEIK 107 variable region amino acid sequence) (without signal sequence)  41 11H9.A1 SYSMN 5 VH CDR1  42 11H9.A1 SISSSSSYIY YADSVKG 17 VH CDR2  43 11H9.A1 LLWFGEIFDY 10 VH CDR3  44 11H9.A1 RASQGISSAL A 11 VL CDR1  45 11H9.A1 DASSLKS 7 VL CDR2  46 11H9.A1 QQFNSYPIT 9 VL CDR3  47 11H9.A1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 50 CH HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP 100 heavy chain KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 150 constant HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 200 region EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 250 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 330  48 11H9.A1 RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 CL NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 light chain SFNRGEC 107 constant region  49 11H9.A1 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS 50 heavy chain ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARLL 100 (without WFGEIFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD 150 signal YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY 200 sequence) ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK 250 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV 350 YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 400 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 449  50 11H9.A1 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 light chain ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPITFGQ 100 (without GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 signal DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 sequence) LSSPVTKSFN RGEC 214  51 2G6.C2 VH GAGGTGCAAC TGGTGGAGTC TGGGGGAGGC TTGGTACAGC GGGGGGGGTC 50 (heavy chain CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT AGCAATAGCA 100 variable TGAACTGGAT CCGCCAGGCT CCAGGGAAGG GGCTGGAGTG GGTTGCACAC 150 region ATTAGTAGTG GTAGCAGTTT CATATACTAC GCAGACTCTG TGAAGGGCCG 200 nucleotide ATTCACCATC TCCAGAGACA ATGCCAAGAA CTCTCTGTCT CTGCAAATGA 250 sequence) ACAGCCTGAG AGACGAAGAC ACGGCTGTGT ATTACTGTGC GAGAGATGAC 300 TGGGGAATTG ATGCTTTTAA TATCTGGGGC CAAGGGACAA TGGTCACCGT 350 CTCTTCA 357  52 2G6.C2 VH EVQLVESGGG LVQRGGSLRL SCAASGFTFS SNSMNWIRQA PGKGLEWVAH 50 (heavy chain ISSGSSFIYY ADSVKGRFTI SRDNAKNSLS LQMNSLRDED TAVYYCARDD 100 variable WGIDAFNIWG QGTMVTVSS 119 region amino acid sequence)  53 2G6.C2VL GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA 50 (light chain AAGAGCCACC CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTCCTTAG 100 variable CCTGGTACCA ACAGAAACCT GGCCAGGCTC CCAGGCTCCT CATCTATGAT 150 region GCATCCAACA GGGCCACTGG CATCCCAGCC AGGTTCAGTG GCAGTGGGTC 200 nucleotide TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT GAAGATTTTG 250 sequence) CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGATATTCAC TTTCGGCCCT 300 GGGACCAAAG TGGATATCAA A 321  54 2G6.C2 VL EIVLTQSPAT LSLSPGERAT LSCRASQSVS SSLAWYQQKP GQAPRLLIYD 50 (light chain ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWIFTFGP 100 variable GTKVDIK 107 region amino acid sequence)  55 2G6.C2 VH SNSMN 5 CDR1  56 2G6.C2 VH HISSGSSFIY YADSVKG 107 CDR2  57 2G6.C2 VH DDWGIDAFNI 10 CDR3  58 2G6.C2 VL RASQSVSSSL A 11 CDR1  59 2G6.C2 VL DASNRAT 7 CDR2  60 2G6.C2 VL QQRSNWIFT 9 CDR3  61 2G6.C2 CH ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 50 heavy chain HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP 100 constant KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 150 region HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 200 EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 250 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 330  62 2G6.C2 CL RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 light chain NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 constant SFNRGEC 107 region  63 2G6.C2 EVQLVESGGG LVQRGGSLRL SCAASGFTFS SNSMNWIRQA PGKGLEWVAH 50 heavy chain ISSGSSFIYY ADSVKGRFTI SRDNAKNSLS LQMNSLRDED TAVYYCARDD 100 WGIDAFNIWG QGTMVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD 150 YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY 200 ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK 250 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV 350 YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 400 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 459  64 2G6.C2 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SSLAWYQQKP GQAPRLLIYD 50 light chain ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWIFTFGP 100 GTKVDIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 LSSPVTKSFN RGEC 214  65 4G5.D1 ATGAAACACC TGTGGTTCTT CCTCCTCCTG GTGGCAGCTC CCAGATGGGT -57 VH (heavy CCTGTCC -7 chain CAGATGCAGC TGCAGGAGTC GGGCCCAGGA CTGGTGAAGC CTTCGGAGAC 50 variable CCTGTCCCTC ACCTGCACTG TCTCTGGTGG CTCCGTCAGC AGTGGTCGTT 100 region ACTACTGGAG TTGGATCCGG CAGCCCCCCG GGAAGGGACT GGAGTGGATT 150 nucleotide GGGTATATCT ATTACAGTGG GAGCACCAAC TACAACCCCT CCCTCAAGAG 200 sequence) TCGAGTCACC ATATCAGTAG ACACGTCCAA GAACCAGTTC TCCCTGAAGC 250 (with 57- TGACCTCTGT GACCGCTGCG GACACGGCCG TGTATTACTG TGCGAGAGAG 300 nucleotide GGTGGAGACT ACTACTACTA CAATATGGAC GTCTGGGGCC CAGGGACCAC 350 signal GGTCACCGTC TCCTCA 367 sequence) 174 4G5.D1 CAGATGCAGC TGCAGGAGTC GGGCCCAGGA CTGGTGAAGC CTTCGGAGAC 50 VH CCTGTCCCTC ACCTGCACTG TCTCTGGTGG CTCCGTCAGC AGTGGTCGTT 100 nucleotide ACTACTGGAG TTGGATCCGG CAGCCCCCCG GGAAGGGACT GGAGTGGATT 150 sequence GGGTATATCT ATTACAGTGG GAGCACCAAC TACAACCCCT CCCTCAAGAG 200 (without TCGAGTCACC ATATCAGTAG ACACGTCCAA GAACCAGTTC TCCCTGAAGC 250 signal TGACCTCTGT GACCGCTGCG GACACGGCCG TGTATTACTG TGCGAGAGAG 300 sequence) GGTGGAGACT ACTACTACTA CAATATGGAC GTCTGGGGCC CAGGGACCAC 350 GGTCACCGTC TCCTCA 367  66 4G5.D1 MKHLWFFLLL VAAPRWVLS -19 VH amino acid QMQLQESGPG LVKPSETLSL TCTVSGGSVS SGRYYWSWIR QPPGKGLEWI 50 sequence GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF SLKLTSVTAA DTAVYYCARE 100 (with 19- GGDYYYYNMD VWGPGTTVTV SS 122 amino acid signal sequence) 175 4G5.D1 QMQLQESGPG LVKPSETLSL TCTVSGGSVS SGRYYWSWIR QPPGKGLEWI 50 VH GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF SLKLTSVTAA DTAVYYCARE 100 amino acid GGDYYYYNMD VWGPGTTVTV SS 122 sequence (without signal sequence)  67 4G5.D1 ATGGAAACCC CAGCGCAGCT TCTCTTCCTC CTGCTACTCT GGCTCCCAGA -60 VL TACCACCGGA -10 nucleotide sequence GAAATTGTGT TGACGCAGTC TCCAGGCACC CTGTCTTTGT CTCCAGGGGA 50 (with 60- AAGAGCCACC CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCAGCTACT 100 nucleotide TAGCCTGGTA CCAGCAGAAA CCTGGCCAGG CTCCCAGGCT CCTCATCTAT 150 signal GGTGCATCCA GCAGGGCCAC TGGCATCCCA GACAGGTTCA GTGGCAGTGG 200 sequence) GTCTGGGACA GACTTCACTC TCACCATCAG CAGACTGGAG CCTGAAGATT 250 TTGCAGTGTA TTACTGTCAG CAGTATGGTA GCTCACCGTA CACTTTTGGC 300 CAGGGGACCA AGCTGGAGAT CAAA 324 176 4G5.D1 GAAATTGTGT TGACGCAGTC TCCAGGCACC CTGTCTTTGT CTCCAGGGGA 50 VL AAGAGCCACC CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCAGCTACT 100 nucleotide TAGCCTGGTA CCAGCAGAAA CCTGGCCAGG CTCCCAGGCT CCTCATCTAT 150 sequence GGTGCATCCA GCAGGGCCAC TGGCATCCCA GACAGGTTCA GTGGCAGTGG 200 (without GTCTGGGACA GACTTCACTC TCACCATCAG CAGACTGGAG CCTGAAGATT 250 signal TTGCAGTGTA TTACTGTCAG CAGTATGGTA GCTCACCGTA CACTTTTGGC 300 sequence) CAGGGGACCA AGCTGGAGAT CAAA 324  68 4G5.D1 METPAQLLFL LLLWLPDTTG -20 VL amino EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY 50 acid GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPYTFG 100 sequence QGTKLEIK 108 (with 20- amino acid signal sequence) 177 4G5.D1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY 50 VL amino GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPYTFG 100 acid QGTKLEIK 108 sequence (without signal sequence)  69 4G5.D1 SGRYYWS 8 VH CDR1  70 4G5.D1 YIYYSGSTNY NPSLKS 16 VH CDR2  71 4G5.D1 EGGDYYYYNM DV 12 VH CDR3  72 4G5.D1 RASQSVSSSY LA 12 VL CDR1  73 4G5.D1 GASSRAT 7 VL CDR2  74 4G5.D1 QQYGSSPYT 9 VL CDR3  75 4G5.D1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 50 CH heavy HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP 100 chain KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 150 constant HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 200 region EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 250 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 330  76 4G5.D1 RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 CL NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 light chain SFNRGEC 107 constant region  77 4G5.D1 QMQLQESGPG LVKPSETLSL TCTVSGGSVS SGRYYWSWIR QPPGKGLEWI 50 heavy chain GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF SLKLTSVTAA DTAVYYCARE 100 GGDYYYYNMD VWGPGTTVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL 150 VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT 200 QTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELL GGPSVFLFPP 250 KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 300 YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE 350 PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 400 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP 450 GK 452  78 4G5.D1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY 50 light chain GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPYTFG 100 QGTKLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK 150 VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ 200 GLSSPVTKSF NRGEC 215  79 1G5.B2 ATGGAGTTTG GGCTGAGCTG GGTTTTCCTC GTTGCTCTTT TAAGAGGTGT -57 VH CCAGTGT -7 nucleotide CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC GTGGTCCAGC CTGGGAGGTC 50 sequence CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT GACTATGCTA 100 (with 57- TGCACTGGGT CCGCCAGACT CCAGGCAGGG GGCTGGAGTG GCTGACATTT 150 nucleotide ATATCATATG ATGGAAGCAA TAAATACCAC GCAGACTCCG TGAAGGGCCG 200 signal ATTCACCATC TCCAGAGACA ATTCCAAGAA CACGCTGTTT CTGCAAATGA 250 sequence) ACAGTCTGAG AGCTGAGGAC ACGGCTGTTT ATTACTGTGC GAGAGATTCC 300 TGGGATCGGG GGTACTTCGA TCTCTGGGGC CGTGGCACCC TGGTCACTGT 350 CTCCTCA 357 178 1G5.B2 CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC GTGGTCCAGC CTGGGAGGTC 50 VH CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT GACTATGCTA 100 nucleotide TGCACTGGGT CCGCCAGACT CCAGGCAGGG GGCTGGAGTG GCTGACATTT 150 sequence ATATCATATG ATGGAAGCAA TAAATACCAC GCAGACTCCG TGAAGGGCCG 200 (without ATTCACCATC TCCAGAGACA ATTCCAAGAA CACGCTGTTT CTGCAAATGA 250 signal ACAGTCTGAG AGCTGAGGAC ACGGCTGTTT ATTACTGTGC GAGAGATTCC 300 sequence) TGGGATCGGG GGTACTTCGA TCTCTGGGGC CGTGGCACCC TGGTCACTGT 350 CTCCTCA 357  80 1G5.B2 MEFGLSWVFL VALLRGVQC -19 VH QVQLVESGGG VVQPGRSLRL SCAASGFTFS DYAMHWVRQT PGRGLEWLTF 50 amino acid ISYDGSNKYH ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCARDS 100 sequence WDRGYFDLWG RGTLVTVSS 119 (with 19- amino acid signal sequence) 179 1G5.B2 QVQLVESGGG VVQPGRSLRL SCAASGFTFS DYAMHWVRQT PGRGLEWLTF 50 VH ISYDGSNKYH ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCARDS 100 amino acid WDRGYFDLWG RGTLVTVSS 119 sequence (without signal sequence)  81 1G5.B2 ATGGAAGCCC CAGCTCAGCT TCTCTTCCTC CTGCTACTCT GGCTCCCAGA -60 VL TACCACCGGA -10 nucleotide GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA 50 sequence AAGAGCCACC CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG 100 (with 60- CCTGGTACCA ACAGAAACCT GGCCAGGCTC CCAGGCTCCT CATCTATGAT 150 nucleotide GCATCCAACA GGGCCACTGG CATCCCAGCC AGGTTCAGTG GCAGTGGGTC 200 signal TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT GAAGATTTTG 250 sequence) CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGTGGACGTT CGGCCAAGGG 300 ACCAAGGTGG AAATCAAA 318 180 1G5.B2 GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA 50 VL AAGAGCCACC CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG 100 nucleotide CCTGGTACCA ACAGAAACCT GGCCAGGCTC CCAGGCTCCT CATCTATGAT 150 sequence GCATCCAACA GGGCCACTGG CATCCCAGCC AGGTTCAGTG GCAGTGGGTC 200 (without TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT GAAGATTTTG 250 signal CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGTGGACGTT CGGCCAAGGG 300 sequence) ACCAAGGTGG AAATCAAA 318  82 1G5.B2 MEAPAQLLFL LLLWLPDTTG -20 VL amino EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD 50 acid ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWWTFGQG 100 sequence TKVEIK 106 (with 20- amino acid signal sequence) 181 1G5.B2 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD 50 VL amino ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWWTFGQG 100 acid TKVEIK 106 sequence (without signal sequence)  83 1G5.B2 DYAMH 5 VH CDR1  84 1G5.B2 FISYDGSNKY HADSVKG 17 VH CDR2  85 1G5.B2 DSWDRGYFDL 10 VH CDR3  86 1G5.B2 RASQSVSSYL A 11 VL CDR1  87 1G5.B2 DASNRAT 7 VL CDR2  88 1G5.B2 QQRSNWWT 8 VL CDR3  89 1G5.B2 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 50 CH heavy HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP 100 chain KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 150 constant HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 200 region EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 250 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 330  90 1G5.B2 RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 CL NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 light chain SFNRGEC 107 constant region  91 1G5.B2 QVQLVESGGG VVQPGRSLRL SCAASGFTFS DYAMHWVRQT PGRGLEWLTF 50 heavy chain ISYDGSNKYH ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCARDS 100 (without WDRGYFDLWG RGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD 150 signal YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY 200 sequence) ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK 250 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV 350 YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 400 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 449  92 1G5.B2 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD 50 light chain ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWWTFGQG 100 (without TKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD 150 signal NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL 200 sequence) SSPVTKSFNR GEC 213  93 P1- QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYAMHWVRQA PGKGLEWVAV 50 069366 ISYDGSYKEY ADSVKGRFTI SRDSSKNTLY LQMNSLRAED TAVYYCARAQ 100 VH ISEYFDYWGQ GTLVTVSS 118 (heavy chain variable region amino acid sequence)  94 P1- DIQMTQSPSS LSASVGDRVT ITCRASQGIS SWLAWYQQKP EKAPKSLIYA 50 069366 ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNSYPLTFGQ 100 VL GTKVEIK 107 (light chain variable region amino acid sequence)  95 P1- SYAMH 5 069366 VH CDR1  96 P1- VISYDGSYKEYADSVKG 069366 VH CDR2  97 P1- AQISEYFDY 069366 VH CDR3  98 P1- RASQGISSWLA 069366 VL CDR1  99 P1- AASSLQS 069366 VL CDR2 100 P1- QQYNSYPLT 069366 VL CDR3 101 P1- ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 50 069366 HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP 100 CH heavy KSCDKTHTCP PCPAPEAEGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 150 chain HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 200 constant EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC 250 region LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEALHNHYT QKSLSLSPG 329 102 P1- RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 069366 NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 CL SFNRGEC 107 light chain constant region 103 P1- QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYAMHWVRQA PGKGLEWVAV 50 069366 ISYDGSYKEY ADSVKGRFTI SRDSSKNTLY LQMNSLRAED TAVYYCARAQ 100 heavy chain ISEYFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 without FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 signal CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 sequence TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPG 447 104 P1- DIQMTQSPSS LSASVGDRVT ITCRASQGIS SWLAWYQQKP EKAPKSLIYA 50 069366 ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNSYPLTFGQ 100 light chain GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 without DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 signal LSSPVTKSFN RGEC 214 sequence 105 27H4.D4 CAGGTGCAAC TAGTGGAGTC TGGGGGAGGC TTGGTCAAGC CTGGAGGGTC 50 VH CCTCAGACTC TCCTGTGCAG CCTCTGGGTT CACCTTCAGT GACTTCTACA 100 (heavy chain TGAGCTGGAT CCGCCAGGCT CCAGGGAAGG GGCTGGAGTG GATTTCATAC 150 variable ATTAGTAGTA GTGATTTTAC CATATACTAC GCAGACTCTG TGGAGGGCCG 200 region ATTCACCATC TCCAGGGACA ACGCCAAGAA CTCACTGTTT CTGCAAATGA 250 nucleotide ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAAGAGGG 300 sequence) AGCCTTCCTT TCAACTACGA TATGGACGTC TGGGGCCAAG GGACCACGGT 350 CACCGTCTCC TCA 363 106 27H4.D4 QVQLVESGGG LVKPGGSLRL SCAASGFTFS DFYMSWIRQA PGKGLEWISY 50 VH ISSSDFTIYY ADSVEGRFTI SRDNAKNSLF LQMNSLRAED TAVYYCARRG 100 (heavy chain SLPFNYDMDV WGQGTTVTVS S 121 variable region amino acid sequence) 107 27H4.D4 GACATCCAGA TGACCCAGTC TCCATCTTCC GTGTCTGCAT CTGTAGGAGA 50 VL CAGAGTCACC ATCTCTTGTC GGGCGAGTCA GGGTATTAGC AGCTACTTAG 100 (light chain CCTGGTATCA GCATAAACCA GGGAAAGCCC CTAAGCTCCT GATCTATGCT 150 variable GCATCCAGTT TGCAAAGTGG GGTCCCATCA AGGTTCAGCG GCAGTGGATC 200 region TGGGACAGCT TTCACTCTCA CCATCAGCAG CCTGCAGCCT GAAGATTTTG 250 nucleotide CAACTTACTA TTGTCAACAG GCTAATAGTT TCCCGCTCAC TTTCGGCGGA 500 sequence) GGGACCAAGG TGGAGATCAA A 521 108 27H4.D4 DIQMTQSPSS VSASVGDRVT ISCRASQGIS SYLAWYQHKP GKAPKLLIYA 50 VL ASSLQSGVPS RFSGSGSGTA FTLTISSLQP EDFATYYCQQ ANSFPLTFGG 100 (light chain GTKVEIK 107 variable region amino acid sequence) 109 27H4.D4 DFYMS 5 VH CDR1 110 27H4.D4 YISSSDFTIY YADSVEG 17 VH CDR2 111 27H4.D4 RGSLPFNYDM DV 12 VH CDR3 112 27H4.D4 RASQGISSYL A 11 VL CDR1 113 27H4.D4 AASSLQS 7 VL CDR2 114 27H4.D4 QQANSFPLT 9 VL CDR3 115 27H4.D4 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 50 CH heavy HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP 100 chain KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 150 constant HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 200 region EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 250 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 330 116 27H4.D4 RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 CL NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 light chain SFNRGEC 107 constant region 117 27H4.D4 QVQLVESGGG LVKPGGSLRL SCAASGFTFS DFYMSWIRQA PGKGLEWISY 50 heavy chain ISSSDFTIYY ADSVEGRFTI SRDNAKNSLF LQMNSLRAED TAVYYCARRG 100 without SLPFNYDMDV WGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150 signal KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ 200 sequence TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK 250 PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300 NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP 350 QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450 K 451 118 27H4.D4 DIQMTQSPSS VSASVGDRVT ISCRASQGIS SYLAWYQHKP GKAPKLLIYA 50 light chain ASSLQSGVPS RFSGSGSGTA FTLTISSLQP EDFATYYCQQ ANSFPLTFGG 100 without GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 signal DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 sequence LSSPVTKSFN RGEC 214 126 NKG2A.10 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 heavy ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 chain WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 FPEPVTVSWN SGALTSGVHT FPA 173 127 NKG2A.10 AIQLTQSPSS LSASVGDRVT ITCRASQGIQ SALAWYQQKP GKAPKLLIYD 50 light ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 LSSPVTKSFN RGEC 214 128 NKG2A.12 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 heavy ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 chain WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 129 NKG2A.12 AIQLTQSPSS LSASVGDRVT ITCRASQGIN SALAWYQQKP GKAPKLLIYD 50 light ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 LSSPVTKSFN RGEC 214 130 NKG2A.14 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 heavy ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 chain WGLPFDYWGQ GILVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 131 NKG2A.14 AIQLTQSPSS LSASVGDRVT ITCRASQGIQ SALAWYQQKP GKAPKLLIYD 50 light ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 LSSPVTKSFN RGEC 214 132 NKG2A.15 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 heavy ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 chain WGLPFDYWGQ GILVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 133 NKG2A.15 AIQLTQSPSS LSASVGDRVT ITCRASQGIP SALAWYQQKP GKAPKLLIYD 50 light ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 LSSPVTKSFN RGEC 214 134 NKG2A.18 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 VH ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 (heavy chain WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 variable FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 region CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 nucleotide TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 sequence) YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 135 NKG2A.18 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLISD 50 LH ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 (light chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 variable DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 region LSSPVTKSFN RGEC 214 nucleotide sequence) 136 NKG2A.19 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 VH ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 (heavy chain WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 variable FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 region CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 nucleotide TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 sequence) YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 137 NKG2A.19 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 LH ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSTPLTFGQ 100 (light chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 variable DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 region LSSPVTKSFN RGEC 214 nucleotide sequence) 138 NKG2A.20 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 VH ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 (heavy chain WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 variable FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 region CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 nucleotide TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 sequence) YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 139 NKG2A.20 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 VH ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSAPLTFGQ 100 (light chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 variable DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 region LSSPVTKSFN RGEC 214 nucleotide sequence) 140 NKG2A.21 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 VH ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 (heavy chain WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 variable FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 region CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 nucleotide TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 sequence) YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 141 NKG2A.21 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 LH ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSNPLTFGQ 100 (light chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 variable DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 region LSSPVTKSFN RGEC 214 nucleotide sequence) 142 NKG2A.22 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 VH ISSSSSTIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 (heavy chain WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 variable FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 region CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 nucleotide TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 sequence) YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 143 NKG2A.22 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 LH ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 (light chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 variable DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 region LSSPVTKSFN RGEC 214 nucleotide sequence) 144 NKG2A.23 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 VH ISSSSSYTYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 (heavy chain WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 variable FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 region CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 nucleotide TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 sequence) YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 145 NKG2A.23 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 LH ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 (light chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 variable DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 region LSSPVTKSFN RGEC 214 nucleotide sequence) 146 NKG2A.24 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 VH ISSSSSYINY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 (heavy chain WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 variable FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 region CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 nucleotide TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 sequence) YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 147 NKG2A.24 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 LH ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 (light chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 variable DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 region LSSPVTKSFN RGEC 214 nucleotide sequence) 148 NKG2A.25 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 VH ISSSSSYISY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 (heavy chain WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 variable FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 region CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 nucleotide TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 sequence) YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 149 NKG2A.25 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 LH ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 (light chain GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 variable DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 region LSSPVTKSFN RGEC 214 nucleotide sequence) 150 NKG2A.13- AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 G1.3f ASSLKSGVPS FSGSGSGTDF TLTISSLQPE DFATYYCQQF NSYPLTFGQG 100 (light chain TRLEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD 150 sequence) NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL 200 SSPVTKSFNR GEC 213 151 NKG2A.13- EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 G1.3f ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 (heavy chain WGLPFDYWGQ GILVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 sequence) FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 152 mNKG2A.3- DIVMTQSPSS LAVSAGDKVT INCKSSQTLF SGRYNYLAWY QQKTGQAPKL 50 mG1- LIYYTSTRHT GVPGRFIGSG SGTDFTLTIN NLQTEDLGNY YCQQHYSTPY 100 D265A TFGAGTNLEI RRADAAPTVS IFPPSSEQLT SGGASVVCFL NNFYPKDINV 150 (Light chain KWKIDGSERQ NGVLNSWTDQ DSKDSTYSMS STLTLTKDEY ERHNSYTCEA 200 sequence) THKTSTSPIV KSFNRNEC 218 153 mNKG2A.3- QVQLKQSGAE LVKPGASVKI SCKTSGYTFT DGYMHWVEQN PGQGLEWIGR 50 mG1- IDPDSGYTMY NQKFQDKATL TRDKSSSTVY MELRSLTSED SAVYYCAINY 100 D265A GEYWYFDFWG QGTQVTVSSA KTTPPSVYPL APGSAAQTNS MVTLGCLVKG 150 (Heavy YFPEPVTVTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVP SSTWPSETVT 200 chain CNVAHPASST KVDKKIVPRD CGCKPCICTV PEVSSVFIFP PKPKDVLTIT 250 sequence) LTPKVTCVVV AISKDDPEVQ FSWFVDDVEV HTAQTQPREE QFNSTFRSVS 300 ELPIMHQDWL NGKEFKCRVN SAAFPAPIEK TISKTKGRPK APQVYTIPPP 350 KEQMAKDKVS LTCMITDFFP EDITVEWQWN GQPAENYKNT QPIMDTDGSY 400 FVYSKLNVQK SNWEAGNTFT CSVLHEGLHN HHTEKSLSHS PGK 443 154 RASQGIPSAL A 11 155 RASQGINSAL A 11 156 LSIDNEEMKF 10 157 PSSWIGVFRN SSHHPW 16 158 LAFKHEIKDS DN 12 159 QVNRLKSAQQ CGSSIIYHC 19 160 GGGGSGGGGS GGGGS 15 161 PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT 50 ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET 100 TFMCEYADET ATIVEFLNRW ITFSQSIIST LT 132 162 VSNK 4 163 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 164 AIQLTQSPSS LSASVGDRVT ITCRASQGIP SALAWYQQKP GKAPKLLIYD 50 ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 GTRLEIK 107 165 RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 SFNRGEC 107 166 GAGGTGCAGC TGGTGGAGTC TGGGGGAGGC CTGGTCAAGC CGGGGGGGTC 50 CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT TCCCATAGTA 100 TGAACTGGGT CCGCCAGGCT CCAGGGAAGG GGCTGGAGTG GGTCTCAGCC 150 ATAAGTAGTA GTAGTAGTTA CATATACTAC GCAGACTCAG TGAAGGGCCG 200 ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTAT CTGCAAATGA 250 ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAAGAG 300 TGGGGGCTAC CCTTTGACTA CTGGGGCCAG GGAATCCTGG TCACCGTCTC 350 CTCA 354 167 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 WGLPFDYWGQ GILVTVSS 118 168 GCCATCCAGT TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA 50 CAGAGTCACC ATCACTTGCC GGGCAAGTCA GGGCATTAAC AGTGCTTTAG 100 CCTGGTATCA GCAGAAACCA GGGAAAGCTC CTAAGCTCCT GATCTATGAT 150 GCCTCCAGTT TGAAAAGTGG GGTCCCATCA AGGTTCAGCG GCAGTGGATC 200 TGGGACAGAT TTCACTCTCA CCATCAGCAG CCTGCAGCCT GAAGATTTTG 250 CAACTTATTA CTGTCAACAG TTTAATAGTT ACCCTCTCAC CTTCGGCCAA 300 GGGACACGAC TGGAGATTAA A 321 169 AIQLTQSPSS LSASVGDRVT ITCRASQGIN SALAWYQQKP GKAPKLLIYD 50 ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 GTRLEIK 107 170 GAGGTGCAGT TGGTGGAGTC TGGGGGAGGC CTGGTCAAGC CTGGGGGGTC 50 CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT AGCTATAGCA 100 TGAACTGGGT CCGCCAGGCT CCAGGGAAGG GGCTGGAGTG GGTCTCATCC 150 ATTAGTAGTA GTAGTAGTTA CATATACTAC GCAGACTCAG TGAAGGGCCG 200 ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTAT CTGCAAATGA 250 ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGACTACTA 300 TGGTTCGGGG AGATTTTTGA CTACTGGGGC CAGGGAACCC TGGTCACCGT 350 CTCCTCA 357 171 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS 50 ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARLL 100 WFGEIFDYWG QGTLVTVSS 119 172 GCCATCCAGT TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGAGA 50 CAGAGTCACC ATCACTTGCC GGGCAAGTCA GGGCATTAGC AGTGCTTTAG 100 CCTGGTATCA GCAGAAACCA GGGAAAGCTC CTAAGCTCCT GATCTATGAT 150 GCCTCCAGTT TGAAAAGTGG GGTCCCATCA AGGTTCAGCG GCAGTGGATC 200 TGGGACAGAT TTCACTCTCA CCATCAGCAG CCTGCAGCCT GAAGATTTTG 250 CAACTTATTA CTGTCAACAG TTTAATAGTT ACCCGATCAC CTTCGGCCAA 300 GGGACACGAC TGGAGATTAA A 321 173 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPITFGQ 100 GTRLEIK 107 174 CAGATGCAGC TGCAGGAGTC GGGCCCAGGA CTGGTGAAGC CTTCGGAGAC 50 CCTGTCCCTC ACCTGCACTG TCTCTGGTGG CTCCGTCAGC AGTGGTCGTT 100 ACTACTGGAG TTGGATCCGG CAGCCCCCCG GGAAGGGACT GGAGTGGATT 150 GGGTATATCT ATTACAGTGG GAGCACCAAC TACAACCCCT CCCTCAAGAG 200 TCGAGTCACC ATATCAGTAG ACACGTCCAA GAACCAGTTC TCCCTGAAGC 250 TGACCTCTGT GACCGCTGCG GACACGGCCG TGTATTACTG TGCGAGAGAG 300 GGTGGAGACT ACTACTACTA CAATATGGAC GTCTGGGGCC CAGGGACCAC 350 GGTCACCGTC TCCTCA 366 175 QMQLQESGPG LVKPSETLSL TCTVSGGSVS SGRYYWSWIR QPPGKGLEWI 50 GYIYYSGSTN YNPSLKSRVT ISVDTSKNQF SLKLTSVTAA DTAVYYCARE 100 GGDYYYYNMD VWGPGTTVTV SS 122 176 GAAATTGTGT TGACGCAGTC TCCAGGCACC CTGTCTTTGT CTCCAGGGGA 50 AAGAGCCACC CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCAGCTACT 100 TAGCCTGGTA CCAGCAGAAA CCTGGCCAGG CTCCCAGGCT CCTCATCTAT 150 GGTGCATCCA GCAGGGCCAC TGGCATCCCA GACAGGTTCA GTGGCAGTGG 200 GTCTGGGACA GACTTCACTC TCACCATCAG CAGACTGGAG CCTGAAGATT 250 TTGCAGTGTA TTACTGTCAG CAGTATGGTA GCTCACCGTA CACTTTTGGC 300 CAGGGGACCA AGCTGGAGAT CAAA 324 177 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY 50 GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPYTFG 100 QGTKLEIK 108 178 CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC GTGGTCCAGC CTGGGAGGTC 50 CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT GACTATGCTA 100 TGCACTGGGT CCGCCAGACT CCAGGCAGGG GGCTGGAGTG GCTGACATTT 150 ATATCATATG ATGGAAGCAA TAAATACCAC GCAGACTCCG TGAAGGGCCG 200 ATTCACCATC TCCAGAGACA ATTCCAAGAA CACGCTGTTT CTGCAAATGA 250 ACAGTCTGAG AGCTGAGGAC ACGGCTGTTT ATTACTGTGC GAGAGATTCC 300 TGGGATCGGG GGTACTTCGA TCTCTGGGGC CGTGGCACCC TGGTCACTGT 350 CTCCTCA 357 179 QVQLVESGGG VVQPGRSLRL SCAASGFTFS DYAMHWVRQT PGRGLEWLTF 50 ISYDGSNKYH ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCARDS 100 WDRGYFDLWG RGTLVTVSS 119 180 GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA 50 AAGAGCCACC CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG 100 CCTGGTACCA ACAGAAACCT GGCCAGGCTC CCAGGCTCCT CATCTATGAT 150 GCATCCAACA GGGCCACTGG CATCCCAGCC AGGTTCAGTG GCAGTGGGTC 200 TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT GAAGATTTTG 250 CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGTGGACGTT CGGCCAAGGG 300 ACCAAGGTGG AAATCAAA 318 181 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD 50 ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWWTFGQG 100 TKVEIK 106 182 MDNQGVIYSD LNLPPNPKRQ QRKPKGNKSS ILATEQEITY AELNLQKASQ 50 DFQGNDKTYH CKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSTLIQR 100 HNNSSLNTRT QKARHCGHCP EEWITYSNSC YYIGKERRTW EESLLACTSK 150 NSSLLSIDNE EEMKFLSIIS PSSWIGVFRN SSHHPWVTMN GLAFKHEIKD 200 SDNAELNCAV LQVNRLKSAQ CGSSIIYHCK HKL 233 183 XEWGLPFD 8 184 EXWGLPFD 8 185 EEXGLPFD 8 186 EEWXLPFD 8 187 EEWGXPFD 8 188 EEWGLXFD 8 189 EEWGLPXD 8 190 EEWGLPFX 8 191 GTFSSYSMN 9 192 GFTESSHSMN 10 193 EEWGLPFD 8 194 RASQGSNSAL A 11 195 QQFNNYPLT 9 196 GFTESSYSMN 10 197 EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 WGLPFDYWGQ GILVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 FPEPVTVSWN 160 198 AIQLTQSPSS LSASVGDRVT ITCRASQGIN SALAWYQQKP GKAPKLLIYD 50 ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYcQQ FNSYPLTFGQ 100 GTRLEI 106 199 LQMNSLRAED TAVYYCAREE WGLPFDYWGQ GILVTVSSAS TKGPSVFPLA 50 PSSKSTSGGT AALGCLVKDY FPEPVTVSWN 80 200 LQMNSLRAED TAVYYCAREE WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA 50 PSSKSTSGGT AALGCLVKDY FPEPVTVSWN 80 201 AIQLTQSPSS LSASVGDRVT ITCRASQGIN SALAWYQQKP GKAPKLLIYD 50 ASSLKSGVPS RFSGSGSGTD FTLTISSLQP 80 202 AIQLTQSPSS LSASVGDRVT ITCRASQGIP SALAWYQQKP GKAPKLLIYD 50 ASSLKSGVPS RFSGSGSGTD FTLTISSLQP 80 203 AIQLTQSPSS LSASVGDRVT ITCRASQGIQ SALAWYQQKP GKAPKLLIYD 50 ASSLKSGVPS RFSGSGSGTD FTLTISSLQP 80 204 AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 ASSLKSGVPS RFSGSGSGTD FTLTISSLQP 80 205 NP_ MDNQGVIYSD LNLPPNPKRQ QRKPKGNKSS ILATEQEITY AELNLQKASQ 50 001291377.1 DFQGNDKTYH CKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSTLIQR 100 HNNSSLNTRT QKARHCGHCP EEWITYSNSC YYIGKERRTW EESLLACTSK 150 NSSLLSIDNE EEMKFLSIIS PSSWIGVFRN SSHHPWVTMN GLAFKHEIKD 200 SDNAELNCAV LQVNRLKSAQ CGSSIIYH 228 206 NP_ MDNQGVIYSD LNLPPNPKRQ QRKPKGNKNS ILATEQEITY AELNLQKASQ 50 015567.2 DFQGNDKTYH CKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSRHCGH 100 CPEEWITYSN SCYYIGKERR TWEESLLACT SKNSSLLSID NEEEMKFLSI 150 ISPSSWIGVF RNSSHHPWVT MNGLAFKHEI KDSDNAELNC AVLQVNRLKS 200 AQCGSSIIYH CKHKL 215 207 NP_ MDNQGVIYSD LNLPPNPKRQ QRKPKGNKSS ILATEQEITY AELNLQKASQ 50 998822.1 DFQGNDKTYH CKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSRHCGH 100 CPEEWITYSN SCYYIGKERR TWEESLLACT SKNSSLLSID NEEEMKFLSI 150 ISPSSWIGVF RNSSHHPWVT MNGLAFKHEI KDSDNAELNC AVLQVNRLKS 200 AQCGSSIIYH CKHKL 215 208 NM_ ACAGTTGAGA GGAGTTTGAG TGGAGATTCA GGGCCATTTT AGTATCTTCT 50 001304448.1 GTAGGACAGA GGTCAGCAAG CATGCCCCAG AGGTACAGAT GTATATGTCT 100 CCCAGGAAGT CTCTGTGGGT GAAGGACTGA TCTCAAGTTG TGGCTGACAC 150 TAGTTAAAGC CAAGTTAGAG GGCTGTTTCA GGGTCTACAT TGAGACTACA 200 GTTGATATGC CTACCTCCTG AGACACTAGT GTGTGAGTCT CCTCCTGGGC 250 CCCTGGGCAA ATGGTTTTGG CAGCATGACC AAGGCCTAAA TGGGGCTGAA 300 GGCAAGCACA GGAGGATGGG TCCCTTTTCA GGTCTGGAGA TGGAATCACT 350 GTTGCTATAG CAGGCCTTTT TATGAGACTA ACCTGGCCTC TCCACTAAAG 400 GATGTGTGAC TTTCTGGGGA CAGAAGAGTA CAGTCCCTGA CATCACACAC 450 TGCAGAGATG GATAACCAAG GAGTAATCTA CTCAGACCTG AATCTGCCCC 500 CAAACCCAAA GAGGCAGCAA CGAAAACCTA AAGGCAATAA AAGCTCCATT 550 TTAGCAACTG AACAGGAAAT AACCTATGCG GAATTAAACC TTCAAAAAGC 600 TTCTCAGGAT TTTCAAGGGA ATGACAAAAC CTATCACTGC AAAGATTTAC 650 CATCAGCTCC AGAGAAGCTC ATTGTTGGGA TCCTGGGAAT TATCTGTCTT 700 ATCTTAATGG CCTCTGTGGT AACGATAGTT GTTATTCCCT CTACATTAAT 750 ACAGAGGCAC AACAATTCTT CCCTGAATAC AAGAACTCAG AAAGCACGTC 800 ATTGTGGCCA TTGTCCTGAG GAGTGGATTA CATATTCCAA CAGTTGTTAC 850 TACATTGGTA AGGAAAGAAG AACTTGGGAA GAGAGTTTGC TGGCCTGTAC 900 TTCGAAGAAC TCCAGTCTGC TTTCTATAGA TAATGAAGAA GAAATGAAAT 950 TTCTGTCCAT CATTTCACCA TCCTCATGGA TTGGTGTGTT TCGTAACAGC 1000 AGTCATCATC CATGGGTGAC AATGAATGGT TTGGCTTTCA AACATGAGAT 1050 AAAAGACTCA GATAATGCTG AACTTAACTG TGCAGTGCTA CAAGTAAATC 1100 GACTTAAATC AGCCCAGTGT GGATCTTCAA TAATATATCA TTAAACTTGT 1150 TAATTTAATA CAATTTACAA CACACCTGC 1179 209 NM_ CCACTCTTGA CTCACTCTGA GCCTTCACAG GGCAGTCTGC GAAGATTGCA 50 002259.5 GGCATTGTTT GTTCTTGTCT TGGATTTATG CCTTTAAATT TCACCTTTTA 100 TTACACAGCT ATAGCAGGCC TTTTTATGAG ACTAACCTGG CCTCTCCACT 150 AAAGGATGTG TGACTTTCTG GGGACAGAAG AGTACAGTCC CTGACATCAC 200 ACACTGCAGA GATGGATAAC CAAGGAGTAA TCTACTCAGA CCTGAATCTG 250 CCCCCAAACC CAAAGAGGCA GCAACGAAAA CCTAAAGGCA ATAAAAACTC 300 CATTTTAGCA ACTGAACAGG AAATAACCTA TGCGGAATTA AACCTTCAAA 350 AAGCTTCTCA GGATTTTCAA GGGAATGACA AAACCTATCA CTGCAAAGAT 400 TTACCATCAG CTCCAGAGAA GCTCATTGTT GGGATCCTGG GAATTATCTG 450 TCTTATCTTA ATGGCCTCTG TGGTAACGAT AGTTGTTATT CCCTCTACAT 500 TAATACAGAG GCACAACAAT TCTTCCCTGA ATACAAGAAC TCAGAAAGCA 550 CGTCATTGTG GCCATTGTCC TGAGGAGTGG ATTACATATT CCAACAGTTG 600 TTACTACATT GGTAAGGAAA GAAGAACTTG GGAAGAGAGT TTGCTGGCCT 650 GTACTTCGAA GAACTCCAGT CTGCTTTCTA TAGATAATGA AGAAGAAATG 700 AAATTTCTGT CCATCATTTC ACCATCCTCA TGGATTGGTG TGTTTCGTAA 750 CAGCAGTCAT CATCCATGGG TGACAATGAA TGGTTTGGCT TTCAAACATG 800 AGATAAAAGA CTCAGATAAT GCTGAACTTA ACTGTGCAGT GCTACAAGTA 850 AATCGACTTA AATCAGCCCA GTGTGGATCT TCAATAATAT ATCATTGTAA 900 GCATAAGCTT TAGAGGTAAA GCGTTTGCAT TTGCAGTGCA TCAGATAAAT 950 TGTATATTTC TTAAAATAGA AATATATTAT GATTGCATAA ATCTTAAAAT 1000 GAATTATGTT ATTTGCTCTA ATAAGAAAAT TCTAAATCAA TTATTGAAAC 1050 AGGATACACA CAATTACTAA AGTACAGACA TCCTAGCATT TGTGTCGGGC 1100 TCATTTTGCT CAACATGGTA TTTGTGGTTT TCAGCCTTTC TAAAAGTTGC 1150 ATGTTATGTG AGTCAGCTTA TAGGAAGTAC CAAGAACAGT CAAACCCATG 1200 GAGACAGAAA GTAGAATAGT GGTTGCCAAT GTCTGAGGGA GGTTGAAATA 1250 GGAGATGACC TCTAACTGAT AGAACGTTAC TTTGTGTCGT GATGAAAACT 1300 TTCTAAATTT CAGTAGTGGT GATGGTTGTA ACTCTGCGAA TATACTAAAC 1350 ATCATTGATT TTTAATCATT TTAAGTGCAT GAAATGTATG CTTTGTACAC 1400 GACACTTCAA TAAAGCTATC CAGAAAAAAA AAAAAA 1436 210 NM_ CCACTCTTGA CTCACTCTGA GCCTTCACAG GGCAGTCTGC GAAGATTGCA 50 0073284 GGCATTGTTT GTTCTTGTCT TGGATTTATG CCTTTAAATT TCACCTTTTA 100 TTACACAGCT ATAGCAGGCC TTTTTATGAG ACTAACCTGG CCTCTCCACT 150 AAAGGATGTG TGACTTTCTG GGGACAGAAG AGTACAGTCC CTGACATCAC 200 ACACTGCAGA GATGGATAAC CAAGGAGTAA TCTACTCAGA CCTGAATCTG 250 CCCCCAAACC CAAAGAGGCA GCAACGAAAA CCTAAAGGCA ATAAAAACTC 300 CATTTTAGCA ACTGAACAGG AAATAACCTA TGCGGAATTA AACCTTCAAA 350 AAGCTTCTCA GGATTTTCAA GGGAATGACA AAACCTATCA CTGCAAAGAT 400 TTACCATCAG CTCCAGAGAA GCTCATTGTT GGGATCCTGG GAATTATCTG 450 TCTTATCTTA ATGGCCTCTG TGGTAACGAT AGTTGTTATT CCCTCACGTC 500 ATTGTGGCCA TTGTCCTGAG GAGTGGATTA CATATTCCAA CAGTTGTTAC 550 TACATTGGTA AGGAAAGAAG AACTTGGGAA GAGAGTTTGC TGGCCTGTAC 600 TTCGAAGAAC TCCAGTCTGC TTTCTATAGA TAATGAAGAA GAAATGAAAT 650 TTCTGTCCAT CATTTCACCA TCCTCATGGA TTGGTGTGTT TCGTAACAGC 700 AGTCATCATC CATGGGTGAC AATGAATGGT TTGGCTTTCA AACATGAGAT 750 AAAAGACTCA GATAATGCTG AACTTAACTG TGCAGTGCTA CAAGTAAATC 800 GACTTAAATC AGCCCAGTGT GGATCTTCAA TAATATATCA TTGTAAGCAT 850 AAGCTTTAGA GGTAAAGCGT TTGCATTTGC AGTGCATCAG ATAAATTGTA 900 TATTTCTTAA AATAGAAATA TATTATGATT GCATAAATCT TAAAATGAAT 950 TATGTTATTT GCTCTAATAA GAAAATTCTA AATCAATTAT TGAAACAGGA 1000 TACACACAAT TACTAAAGTA CAGACATCCT AGCATTTGTG TCGGGCTCAT 1050 TTTGCTCAAC ATGGTATTTG TGGTTTTCAG CCTTTCTAAA AGTTGCATGT 1100 TATGTGAGTC AGCTTATAGG AAGTACCAAG AACAGTCAAA CCCATGGAGA 1150 CAGAAAGTAG AATAGTGGTT GCCAATGTCT GAGGGAGGTT GAAATAGGAG 1200 ATGACCTCTA ACTGATAGAA CGTTACTTTG TGTCGTGATG AAAACTTTCT 1250 AAATTTCAGT AGTGGTGATG GTTGTAACTC TGCGAATATA CTAAACATCA 1300 TTGATTTTTA ATCATTTTAA GTGCATGAAA TGTATGCTTT GTACACGACA 1350 CTTCAATAAA GCTATCCAGA AAAAAAAAAA AA 1382 211  NM_ GCATGCCCCA GAGGTACAGA TGTATATGTC TCCCAGGAAG TCTCTGTGGG 50 213657.2 TGAAGGACTG ATCTCAAGTT GTGGCTGACA CTAGTTAAAG CCAAGTTAGA 100 GGGCTGTTTC AGGGTCTACA TTGAGACTAC AGTTGATATG CCTACCTCCT 150 GAGACACTAG TGTGTGAGTC TCCTCCTGGG CCCCTGGGCA AATGGTTTTG 200 GCAGCATGAC CAAGGCCTAA ATGGGGCTGA AGGCAAGCAC AGGAGGATGG 250 GTCCCTTTTC AGGTCTGGAG ATGGAATCAC TGTTGCTATA GCAGGCCTTT 300 TTATGAGACT AACCTGGCCT CTCCACTAAA GGATGTGTGA CTTTCTGGGG 350 ACAGAAGAGT ACAGTCCCTG ACATCACACA CTGCAGAGAT GGATAACCAA 400 GGAGTAATCT ACTCAGACCT GAATCTGCCC CCAAACCCAA AGAGGCAGCA 450 ACGAAAACCT AAAGGCAATA AAAGCTCCAT TTTAGCAACT GAACAGGAAA 500 TAACCTATGC GGAATTAAAC CTTCAAAAAG CTTCTCAGGA TTTTCAAGGG 550 AATGACAAAA CCTATCACTG CAAAGATTTA CCATCAGCTC CAGAGAAGCT 600 CATTGTTGGG ATCCTGGGAA TTATCTGTCT TATCTTAATG GCCTCTGTGG 650 TAACGATAGT TGTTATTCCC TCACGTCATT GTGGCCATTG TCCTGAGGAG 700 TGGATTACAT ATTCCAACAG TTGTTACTAC ATTGGTAAGG AAAGAAGAAC 750 TTGGGAAGAG AGTTTGCTGG CCTGTACTTC GAAGAACTCC AGTCTGCTTT 800 CTATAGATAA TGAAGAAGAA ATGAAATTTC TGTCCATCAT TTCACCATCC 850 TCATGGATTG GTGTGTTTCG TAACAGCAGT CATCATCCAT GGGTGACAAT 900 GAATGGTTTG GCTTTCAAAC ATGAGATAAA AGACTCAGAT AATGCTGAAC 950 TTAACTGTGC AGTGCTACAA GTAAATCGAC TTAAATCAGC CCAGTGTGGA 1000 TCTTCAATAA TATATCATTG TAAGCATAAG CTTTAGAGGT AAAGCGTTTG 1050 CATTTGCAGT GCATCAGATA AATTGTATAT TTCTTAAAAT AGAAATATAT 1100 TATGATTGCA TAAATCTTAA AATGAATTAT GTTATTTGCT CTAATAAGAA 1150 AATTCTAAAT CAATTATTGA AACAGGATAC ACACAATTAC TAAAGTACAG 1200 ACATCCTAGC ATTTGTGTCG GGCTCATTTT GCTCAACATG GTATTTGTGG 1250 TTTTCAGCCT TTCTAAAAGT TGCATGTTAT GTGAGTCAGC TTATAGGAAG 1300 TACCAAGAAC AGTCAAACCC ATGGAGACAG AAAGTAGAAT AGTGGTTGCC 1350 AATGTCTGAG GGAGGTTGAA ATAGGAGATG ACCTCTAACT GATAGAACGT 1400 TACTTTGTGT CGTGATGAAA ACTTTCTAAA TTTCAGTAGT GGTGATGGTT 1450 GTAACTCTGC GAATATACTA AACATCATTG ATTTTTAATC ATTTTAAGTG 1500 CATGAAATGT ATGCTTTGTA CACGACACTT CAATAAAGCT ATCCAGAAAA 1550 AAAAAAAAAA AAA 1563 212  NM_ GCATGCCCCA GAGGTACAGA TGTATATGTC TCCCAGGAAG TCTCTGTGGG 50 213658.2 TGAAGGACTG ATCTCAAGTT GTGGCTGACA CTAGTTAAAG CCAAGTTAGA 100 GGGCTGTTTC AGGGTCTACA TTGAGACTAC AGTTGATATG CCTACCTCCT 150 GAGACACTAG TGTGTGAGTC TCCTCCTGGG CCCCTGGGCA AATGGTTTTG 200 GCAGCATGAC CAAGGCCTAA ATGGGGCTGA AGGCAAGCAC AGGAGGATGG 250 GTCCCTTTTC AGGTCTGGAG ATGGAATCAC TGTTGCTATA GCAGGCCTTT 300 TTATGAGACT AACCTGGCCT CTCCACTAAA GGATGTGTGA CTTTCTGGGG 350 ACAGAAGAGT ACAGTCCCTG ACATCACACA CTGCAGAGAT GGATAACCAA 400 GGAGTAATCT ACTCAGACCT GAATCTGCCC CCAAACCCAA AGAGGCAGCA 450 ACGAAAACCT AAAGGCAATA AAAGCTCCAT TTTAGCAACT GAACAGGAAA 500 TAACCTATGC GGAATTAAAC CTTCAAAAAG CTTCTCAGGA TTTTCAAGGG 550 AATGACAAAA CCTATCACTG CAAAGATTTA CCATCAGCTC CAGAGAAGCT 600 CATTGTTGGG ATCCTGGGAA TTATCTGTCT TATCTTAATG GCCTCTGTGG 650 TAACGATAGT TGTTATTCCC TCTACATTAA TACAGAGGCA CAACAATTCT 700 TCCCTGAATA CAAGAACTCA GAAAGCACGT CATTGTGGCC ATTGTCCTGA 750 GGAGTGGATT ACATATTCCA ACAGTTGTTA CTACATTGGT AAGGAAAGAA 800 GAACTTGGGA AGAGAGTTTG CTGGCCTGTA CTTCGAAGAA CTCCAGTCTG 850 CTTTCTATAG ATAATGAAGA AGAAATGAAA TTTCTGTCCA TCATTTCACC 900 ATCCTCATGG ATTGGTGTGT TTCGTAACAG CAGTCATCAT CCATGGGTGA 950 CAATGAATGG TTTGGCTTTC AAACATGAGA TAAAAGACTC AGATAATGCT 1000 GAACTTAACT GTGCAGTGCT ACAAGTAAAT CGACTTAAAT CAGCCCAGTG 1050 TGGATCTTCA ATAATATATC ATTGTAAGCA TAAGCTTTAG AGGTAAAGCG 1100 TTTGCATTTG CAGTGCATCA GATAAATTGT ATATTTCTTA AAATAGAAAT 1150 ATATTATGAT TGCATAAATC TTAAAATGAA TTATGTTATT TGCTCTAATA 1200 AGAAAATTCT AAATCAATTA TTGAAACAGG ATACACACAA TTACTAAAGT 1250 ACAGACATCC TAGCATTTGT GTCGGGCTCA TTTTGCTCAA CATGGTATTT 1300 GTGGTTTTCA GCCTTTCTAA AAGTTGCATG TTATGTGAGT CAGCTTATAG 1350 GAAGTACCAA GAACAGTCAA ACCCATGGAG ACAGAAAGTA GAATAGTGGT 1400 TGCCAATGTC TGAGGGAGGT TGAAATAGGA GATGACCTCT AACTGATAGA 1450 ACGTTACTTT GTGTCGTGAT GAAAACTTTC TAAATTTCAG TAGTGGTGAT 1500 GGTTGTAACT CTGCGAATAT ACTAAACATC ATTGATTTTT AATCATTTTA 1550 AGTGCATGAA ATGTATGCTT TGTACACGAC ACTTCAATAA AGCTATCCAG 1600 AAAAAAAAAA AAAAAAA 1617

TABLE 2 V_(H), V_(L), C_(H), C_(L), Heavy Chain, and Light Chain Amino Acid Sequences of NKG2A.9 Monoclonal Antibody (also known as 13F3-VH-I107T-Vk-N30S IgG1.3) (hereinafter referred to as “NKG2A.9 Antibody”) Amino Acid Sequences (SEQ ID NOs) Heavy chain EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 variable domain ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 (V_(H)) WGLPFDYWGQ GTLVTVSS 118 (SEQ ID NO: 8) Light chain AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 variable domain ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 (V_(L)) GTRLEIK 107 (SEQ ID NO: 9) Heavy chain ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 50 constant domain HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP 100 (C_(H)) KSCDKTHTCP PCPAPEAEGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 150 (C-terminal HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 200 lysine (K) is not EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC 250 shown but may LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 be included in QQGNVFSCSV MHEALHNHYT QKSLSLSPG 329 some (SEQ ID NO: 16) embodiments) Light chain RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 50 constant domain NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 100 (C_(L)) SFNRGEC 107 (SEQ ID NO: 17) Heavy Chain EVQLVESGGG LVKPGGSLRL SCAASGFTFS SHSMNWVRQA PGKGLEWVSA 50 (C-terminal ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREE 100 lysine (K) is not WGLPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 shown but may FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 be included in CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAEGAPS VFLFPPKPKD 250 some TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 embodiments) YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 350 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 400 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPG 447 (SEQ ID NO: 7) Light Chain AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYD 50 ASSLKSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGQ 100 GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200 LSSPVTKSFN RGEC 214 (SEQ ID NO: 5)

TABLE 3 Amino Acid Sequences for the Six CDR Domains of NKG2A.9 Antibody Amino Acid Sequences (SEQ ID NOs) VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 SHSMN AISSSSSYI EEWGLPFDY RASQGISSALA DASSLKS QQFNSYPLT (SEQ ID YYADSVKG (SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15) NO: 10) (SEQ ID NO: 11)

TABLE 4 Amino Acid Sequences for the Six CDR Domains of NKG2A.11 Antibody Amino Acid Sequences (SEQ ID NOs) VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 SHSMN AISSSSSYI EEWGLPFDY RASQGIPSALA DASSLKS QQFNSYPLT (SEQ ID YYADSVKG (SEQ ID NO: 12) (SEQ ID NO: 154) (SEQ ID NO: 14) (SEQ ID NO: 15) NO: 10) (SEQ ID NO: 11)

TABLE 5 Amino Acid Sequences for the Six CDR Domains of 13F3.A4 Antibody Amino Acid Sequences (SEQ ID NOs) VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL CDR3 SHSMN AISSSSSYI EEWGLPFDY RASQGINSALA DASSLKS QQFNSYPLT (SEQ ID YYADSVKG (SEQ ID NO: 12) (SEQ ID NO: 155) (SEQ ID NO: 14) (SEQ ID NO: 15) NO: 10) (SEQ ID NO: 11) 

1. An isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human natural killer cell inhibitory receptor group 2A (NKG2A) protein, comprising: (a) a heavy chain variable domain comprising amino acid sequences of SEQ ID NOs: 10, 11, 12, and a light chain variable domain comprising amino acid sequences of SEQ ID NOs: 13, 14, and 15; (b) a heavy chain variable domain comprising amino acid sequences of SEQ ID NOs: 10, 11, and 12, and a light chain variable domain comprising amino acid sequences of SEQ ID NOs: 154, 14, and 15; or (c) a heavy chain variable domain comprising amino acid sequences of SEQ ID NOs: 10, 11, and 12, and a light chain variable domain comprising amino acid sequences of SEQ ID NOs: 155, 14, and
 15. 2. An isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human NKG2A protein, and comprises heavy and light chain variable regions, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 167, and/or (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85% at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 164, or SEQ ID NO:
 169. 3. The antibody, or antigen-binding fragment thereof, of claim 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:
 9. 4. The antibody, or antigen-binding fragment thereof, of claim 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:
 164. 5. The antibody, or antigen-binding fragment thereof, of claim 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 167, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:
 169. 6. An isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human NKG2A protein, wherein the heavy and light chains consist essentially of: (a) the amino acid sequences of SEQ ID NOs: 7 and 5, respectively; (b) the amino acid sequences of SEQ ID NOs: 7 and 19, respectively; or (c) the amino acid sequences of SEQ ID NOs: 35 and 36, respectively.
 7. An isolated monoclonal antibody, or antigen-binding fragment thereof, that competes for binding to NKG2A protein with or binds to the same epitope as any one of claims 1, 2, and
 6. 8. An isolated monoclonal antibody, or antigen-binding fragment thereof, that, when bound to human NKG2A protein, specifically binds to the following amino acid residues as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS): (a) (SEQ ID NO: 156) LSIDNEEMKF; (b) (SEQ ID NO: 157) PSSWIGVFRNSSHHPW; (c) (SEQ ID NO: 158) LAFKHEIKDSDN; and (d) (SEQ ID NO: 159) QVNRLKSAQQCGSSIIYHC,

wherein the antibody, or antigen-binding fragment thereof, blocks the binding of HLA-E to human NKG2A protein.
 9. An isolated monoclonal antibody, or antigen-binding fragment thereof, that, when bound to human NKG2A, specifically binds to the following amino acid residues as determined by HDX-MS and/or fast photochemical oxidation of proteins (FPOP) epitope mapping: (a) (SEQ ID NO: 156) LSIDNEEMKF (b) (SEQ ID NO: 157) PSSWIGVFRNSSHHPW (c) (SEQ ID NO: 158) LAFKHEIKDSDN (d) L; and (e) (SEQ ID NO: 159) QVNRLKSAQQCGSSIIYHC,

wherein the antibody, or antigen-binding fragment thereof, blocks the binding of HLA-E to human NKG2A protein.
 10. The antibody of any one of claim 1, 2, 6, 7, 8, or 9, wherein the antibody is a full-length antibody.
 11. The antibody of claim 10, wherein the full-length antibody is an IgG1 antibody.
 12. The antibody, or antigen-binding fragment thereof, of any one of claim 1, 2, 6, 7, 8, or 9, that is an antigen-binding fragment of the antibody.
 13. The antigen-binding fragment of claim 12, wherein the antigen-binding fragment is a Fab, Fab′, (Fab′)2, Fv, or scFv fragment.
 14. The antibody, or antigen-binding fragment thereof, of any one of claim 1, 2, 6, 7, 8, or 9, wherein the antibody is a human, humanized, or chimeric antibody.
 15. An isolated, full-length monoclonal antibody that binds specifically to human NKG2A protein, wherein: the heavy chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, about 100% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and/or the light chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, about 100% identical, or 100% identical to the amino acid sequence of SEQ ID NO:
 5. 16. An isolated, full-length monoclonal antibody that specifically binds to human NKG2A protein, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 7, and the light chain comprise the amino acid sequence set forth in SEQ ID NO:
 19. 17. An isolated, full-length monoclonal antibody that specifically binds to human NKG2A, wherein the heavy chain consists essentially of the amino acid sequence set forth in SEQ ID NO: 7, and the light chain consists essentially of the amino acid sequence set forth in SEQ ID NO:
 19. 18. An isolated, full-length monoclonal antibody that specifically binds to human NKG2A protein, wherein the heavy chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and the light chain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:
 19. 19. An isolated antibody, or antigen binding fragment thereof, that binds to human NKG2A protein and cross-competes for binding to human NKG2A protein with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises the amino acid sequences set forth in SEQ ID NO: 8, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 9; (b) the VH comprises the amino acid sequences set forth in SEQ ID NO: 8, and the VL comprising the amino acid sequence set forth in SEQ ID NO:164; or (c) the VH comprises the amino acid sequences set forth in SEQ ID NO: 167, and the VL comprising the amino acid sequence set forth in SEQ ID NO:
 169. 20. An isolated monoclonal antibody, or an antigen-binding fragment thereof, wherein the antibody specifically binds human NKG2A protein and exhibits at least one of the following properties: (a) reduces binding and/or interaction of HLA-E to human NKG2A protein; (b) reverses NKG2A-mediated inhibitory signaling; (c) does not bind, or binds with low affinity to, human NKG2C protein; (d) binds to human or cynomolgus NKG2A protein; (e) enhances natural killer cell response; (f) enhances the functional activity of T cells; (g) has reduced binding to human Fc gamma receptor (FcγR); (h) induces and/or enhances an anti-tumor immune response; and/or (i) has low immunogenicity in human subjects.
 21. The antibody, or an antigen-binding fragment thereof, of claim 20, wherein the antibody has one or more of the following properties: (a) has an EC₅₀ value of about 0.6 nM or lower for binding to human NKG2A protein as measured by cell binding assay; (b) has an EC₅₀ value of about 9.0 nM or higher for binding to human NKG2C protein as measured by cell binding assay; (c) has an EC₅₀ value for binding to human NKG2A protein that is about 15-fold less than a second EC₅₀ value for binding to human NKG2C protein; (d) has an IC₅₀ value of about 1.0 nM or lower for reducing the binding and/or interaction of HLA-E to human NKG2A protein as measured by cell blocking assay; (e) binds to human NKG2A protein with a K_(D) of about 0.4 nM or lower as measured by Scatchard analysis; (f) binds to human NKG2A protein with a K_(D) of about 61 nM or lower as measured by surface plasmon resonance; (g) binds to cynomolgus NKG2A protein with a K_(D) of about 1.0 nM or lower as measured by Scatchard analysis; (h) is internalized upon binding to NKG2A-expressing cells; (i) increases interferon-gamma (IFNγ) production; (j) wherein the half-life of the anti-NKG2A antibody:NKG2A protein complex is about 40 seconds or longer; and/or (k) exhibits internationalization with an EC₅₀ value of about 0.5 nM or lower.
 22. The antibody, or antigen-binding fragment thereof, of claim 20 or 21, wherein the antibody reduces binding and/or interaction of HLA-E to human NKG2A protein.
 23. The antibody, or antigen-binding fragment thereof, of claim 22, wherein the antibody binds to human NKG2A protein.
 24. An isolated nucleic acid molecule encoding the heavy and/or light chain variable region of the antibody, or antigen binding fragment thereof, of any one of claim 1, 2, 6, 7, 8, 9, 15, 16, 17, 18, 19, or
 20. 25. The isolated nucleic acid of claim 24, wherein the nucleic acid molecule is complementary DNA (cDNA).
 26. An expression vector comprising the nucleic acid molecule of claim
 25. 27. A host cell transformed with the expression vector of claim
 26. 28. An immunoconjugate comprising the antibody, or antigen-binding fragment thereof, according to any one of claim 1, 2, 6, 7, 8, 9, 15, 16, 17, 18, 19, or 20, linked to an agent.
 29. A method of producing the antibody, or antigen-binding fragment thereof, comprising culturing the host cell of claim 27, wherein the antibody is produced.
 30. The method of claim 29, further comprising recovering the antibody, or antigen-binding fragment thereof, from the host cell.
 31. A bispecific molecule comprising the antibody, or antigen-binding fragment thereof, of any one of claim 1, 2, 6, 7, 8, 9, 15, 16, 17, 18, 19, or 20 linked to a second functional moiety.
 32. A composition comprising the antibody, or antigen-binding fragment thereof, of any one of claim 1, 2, 6, 7, 8, 9, 15, 16, 17, 18, 19, or 20, or the bispecific molecule of claim 31, and a pharmaceutically acceptable carrier.
 33. The composition of claim 32, further comprising an additional therapeutic agent.
 34. The composition of claim 33, wherein the additional therapeutic agent is an anti-PD-1 antibody, anti-PD-L1 antibody, and/or an anti-CTLA-4 antibody.
 35. A method for treating or delaying progression of cancer in a human subject comprising administering to the human subject an effective amount of the antibody of any one of claim 1, 2, 6, 7, 8, 9, 15, 16, 17, 18, 19, or 20, the immunoconjugate of claim 28, the bispecific molecule of claim 31, or the composition of claim 33 or 34, to treat or delay the progression of cancer in the human subject.
 36. The method of claim 35, wherein the cancer is bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, endometrial, cervical, gastric, melanoma, renal, urothelial, glioblastoma multiform, or virus-related cancer.
 37. The method of claim 36, wherein the cancer is cervical, head and neck squamous cell carcinoma, pancreatic, non-small cell lung cancer-adenocarcinoma type, non-small cell lung cancer-squamous cell type, gastric, melanoma, colorectal, endometrial, ovarian, renal cell carcinoma, urothelial carcinoma, breast, small cell lung, glioblastoma mulitforme, prostate cancer, or non-Hodgkin lymphoma.
 38. The method of claim 36 or 37, further comprising administering one or more additional therapeutic agent(s) to the human subject.
 39. The method of claim 38, wherein the one or more additional therapeutic agent(s) is a chemotherapeutic agent, a radiotherapeutic agent, and/or an immunotherapeutic agent.
 40. The method of claim 39, wherein the immunotherapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, and/or an anti-CTLA-4 antibody.
 41. The method of claim 40, wherein the anti-PD-1 antibody is nivolumab, and the anti-CTLA-4 antibody is ipilulimab.
 42. A method of stimulating an immune response in a human subject comprising administering to the human subject an effective amount of the antibody of any one of claim 1, 2, 6, 7, 8, 9, 15, 16, 17, 18, 19, or 20, the immunoconjugate of claim 28, the bispecific molecule of claim 31, or the composition of claim 33, 34, or 35, to stimulate the immune response in the human subject.
 43. The method of claim 42, wherein the human subject has a tumor and an anti-tumor immune response is stimulated.
 44. The method of claim 42, wherein the human subject has a chronic viral infection and an anti-viral immune response is stimulated.
 45. A method of detecting presence of NKG2A protein in a sample comprising contacting the sample with the antibody, or antigen-binding fragment thereof, of any one of claim 1, 2, 6, 7, 8, 9, 15, 16, 17, 18, 19, or 20, under conditions that allow for formation of a complex between the antibody, or antigen-binding fragment thereof, and NKG2A protein, and detecting the formation of the complex.
 46. The method of claim 45, wherein the antibody, or antigen-binding fragment thereof, forms a complex more quickly with NKG2A protein than with NKG2C protein.
 47. The method of claim 46, where the antibody, or antigen-binding fragment thereof, forms a complex 15 more quickly with NKG2A protein than with NKG2C protein. 